Vector delivery-based microbicides

ABSTRACT

A new class of anti-microbial agents and methods for preventing or reducing the risk of sexually transmitted infections and/or diseases is provided. Preferably, these anti-microbial agents are also contraceptive and, thus, also prevent or reduce the risk of unplanned pregnancies. The anti-microbial agents comprise a delivery vector having anti-microbial activity (and preferably contraceptive activity) coupled with a nitric oxide donor moiety.

RELATED APPLICATION

This application is based on, and claims benefit of, U.S. ProvisionalApplication Ser. No. 60/708,960, filed Aug. 17, 2005, and which ishereby incorporated by reference.

FIELD OF THE INVENTION

This invention generally relates to a new class of anti-microbial agentsand methods for preventing or reducing the risk of sexually transmittedinfections and/or diseases. Preferably, these anti-microbial agents arealso contraceptive and, thus, also prevent or reduce the risk ofunplanned pregnancies.

BACKGROUND OF THE INVENTION

In recent years, sexually transmitted diseases have become an increasingmedical problem and concern throughout the world. The HIV/AIDS epidemicover the last decade or so has significantly and dramaticallyunderscored the threat of STDs to the human population. Until there is acure, or at least an effective treatment, the best, and perhaps onlyrealistic, approach to this increasing problem of STDs (especiallyHIV/AIDS) appears to be reducing the risk of transmission of STDs by theSTD-causing organisms and thus reducing the number of individuals whobecome newly infected. Even when treatments or cures become available,prevention of infections in the initial instance will likely remain asthe first line of defense. For economic, medical, and psychologicalreasons, it is preferable to prevent the initial infection rather thantreating, and even curing, individuals with STDs.

At present, education in regard to STDs, their modes of transmission,and so-called “safe-sex” techniques has, at least to some degree in themore developed countries, shown promise in reducing the risks of STDtransmission through sexual activity. Screening of the blood supply hashelped to reduce the risk of transmission of such STD-causing organismsvia blood transfusions and related medical practices. Nonetheless, thespread of such STDs has not been halted to a satisfactory degree even indeveloped countries with active and progressive education programs. Evenwith their known effectiveness in preventing STDs, current safe-sextechniques are not always used, or are not always used properly, formany reasons (e.g., carelessness, lack of knowledge, impropertechniques, cultural barriers, unplanned or spontaneous sexual activity,and the like). Moreover, even when used, safe-sex techniques (exceptperhaps abstinence) are not always effective. For example, condoms aregenerally only about 90 percent effective iii preventing conception whenused alone; in the case of such failures, STD-causing organisms, ifpresent, may pass from one sexual partner to the other,

Various birth control devices—including barrier methods and vaginalcontraceptives—are currently available. Some of these may, in addition,also have a least some degree of anti-STD activity. For example, condomscan help prevent the transmission of STDs so long as they are properlyused and/or they perform properly. Nonoxynol-9, currently one of themost widely used contraceptive agents, is reported, at least in somecases, to reduce the risk of transmission of some STOs. Nonoxynol-9,which is a nonionic detergent with strong surfactant properties, acts,like most other chemical-based contraceptives, by killing or otherwiseimmobilizing spermatozoa (e.g., spermicidal activity). Nonoxynol-9 is apotent cytotoxic agent which tends to nonspecifically disrupt cellmembranes. These properties, however, give rise to some very significantdisadvantages. Because nonoxynol-9 is strongly cytotoxic, it can injurevaginal/cervical epithelial and other cells at concentrations as low asabout 0.0005 percent. Clinical studies have confirmed epithelialdisruption of the vagina and cervix. Nonoxynol-9 also disrupts thenormal vaginal flora which provides a protective mechanism, perhaps bymaintaining a low pH, to guard against the invasion of pathogenicmicrobes. Nonoxynol-9 may also partially dissolve or remove theprotective glycoprotein coating in the vagina. The cytotoxic,flora-disruptive, and glycoprotein-removal effects of nonoxynol-9 canlead to vaginal damage or Injury, including lesions. Some women areespecially sensitive to nonoxynol-9 and manifest these effects with onlyoccasional use. The disruption of these protective mechanisms bynonoxynol-9 can actually increase the risks of STD since the breakdownof the protective mechanisms, and especially the occurrence of lesions,allows STD-causing organisms an easier pathway into the cells. Thus, anyanti-STD activity of the contraceptive may be reduced or even lost(i.e., overwhelmed) by the increased risk of infection due to physicaldamage from the contraceptive. Even if such a contraceptive methodprovided some degree of STD protection, it would, of course, mainly bedirected at heterosexual relationships in which pregnancy was notdesired.

More recently contraceptives having anti-STD activity have becomeavailable. U.S. Pat. No. 5,925,621 (Jul. 20, 1999), U.S. Pat. No.5,932,619 (Aug. 3, 1999), U.S. Pat. No. 6,028,115 (Feb. 22, 2000), andU.S. Pat. No. 6,239,182 (May 29, 2001) provide methods for the reductionof sexual transmitted diseases using inhibitory agents such asphosphorylated hesperidins, sulfonated hesperidins, polystyrenesultanates, substituted benzenesulfonic acid formaldehyde co-polymers,H₂SO₄-modified mandelic acids, and the like.

It would be desirable, therefore, to provide more effectiveanti-microbial agents and methods for preventing or reducing the risk ofsexually transmitted infections and/or diseases; preferably suchanti-microbial agents would also be contraceptive and, thus, prevent orreduce the risk of unplanned pregnancies. It would be desirable if suchanti-microbial agents, whether contraceptive or not, and methods wouldnot interfere with the natural and protective vaginal mechanisms itwould also be desirable if such anti-microbial agents, whethercontraceptive or not, and methods would be relatively easy to use, havesignificantly fewer side effects than currently available methods (i.e.,nonoxynol-9) so that it would more likely be used on a consistent basis,and be effective at lower concentrations. It would also be desirable ifsuch anti-microbial agents, whether contraceptive or not, and methodscould be used in heterosexual, homosexual, and bisexual relationshipsand for a wide range of sexual activities. It would also be desirable ifsuch anti-microbial agents, whether contraceptive or not, and methodscould be implemented by either party to the sexual activity. The presentinvention, as detailed in the present specification, provides suchanti-microbial agents and contraceptive anti-microbial agents andmethods.

SUMMARY OF THE INVENTION

This invention generally relates to improved anti-microbial agents andto methods for preventing STDs and/or reducing the risk of transmissionof such STDs through sexual activity using the improved anti-microbialagents. Preferably, such anti-microbial agents are also contraceptive.The anti-microbial agents comprise a delivery vector havinganti-microbial activity (and preferably contraceptive activity) coupledwith a nitric oxide donor moiety. The method is suitable for use byheterosexual, homosexual, and bisexual individuals to significantlyreduce the risk of being infected by, or of transmitting, a STD throughsexual contact. Moreover, the risk of pregnancy during heterosexualactivity is also significantly reduced in preferred embodiments.Although this method can be used alone, it is generally preferred thatit be used in conjunction with other so-called “safe sex” techniques inorder to even further reduce the risk of STD transmission or Infection.

The method of this invention generally comprises the application of aneffective amount of the improved anti-microbial agent or agents to thearea or areas of sexual contact (e.g., genitalia) of at least one (andpreferably all) of the participants prior to engaging in sexualactivity. The anti-microbial agents of this invention comprises adelivery vector component having anti-microbial activity (and preferablycontraceptive activity) coupled with a nitric oxide donor moiety. Forpurposes of this invention the “anti-microbial agent” is a compound ormixture of compounds which can inactivate at least one major STD-causingorganisms (HIV, HSV, gonococci, papilloma virus, and/or chlamydia)without necessarily killing them and which generates nitric oxide insitu (i.e., at the binding site of the delivery vector component). Thenitric oxide, thus released, can kill or otherwise inactivate themicrobe; the microbe can also be killed or otherwise inactivated by thedelivery vector component. “Anti-microbial agents” of this invention mayor may not (but preferably do) have contraceptive activity in additionto the anti-microbial activity. Vector delivery components which arepreferred in the present invention for preparing anti-microbial agentsare inhibitory agents such as phosphorylated hesperidins, sulfonatedhesperidins, polystyrene sulfonates, substituted benzenesulfonic acidformaldehyde co-polymers, H₂SO₄-modified mandelic acids, cellulosesulfates, and the like. Thus, preferred NO-coupled anti-microbial agentsof this invention include, for example, phosphorylated hesperidinscoupled with a NO-donor, sulfonated hesperidins coupled with a NO-donor,polystyrene sulfonates coupled with a NO-donor, substitutedbenzenesulfonic acid formaldehyde co-polymers coupled with a NO-donor,H₂SO₄-modified mandelic acids coupled with a NO-donor, and cellulosesulfates coupled with a NO-donor, and the like. Preferably the vectordelivery components as well as the NO-coupled anti-microbial agents usedin the present invention are at least partially water soluble or waterdispersable so that anti-SW formulations can more easily be prepared.More preferred anti-microbial agents for use in this invention includeH₂SO₄-modified mandelic acids (SAMMAs) coupled with a NO-donor (i.e.,NO-SAMMAs). Preferred NO-SAMMAs include those which have beenfractionated so as to have a narrower molecular weight distribution andincreased activities.

In addition to anti-STD activity, these compounds may also act asvaginal contraceptives (and preferably do act as such) and generallyhave fewer side effects than conventional vaginal contraceptives (e.g.,nonoxynol-9). For example, the compounds useful in this invention aregenerally not toxic (or only minimally toxic) to natural and beneficialvaginal flora and, thus, do not significantly upset the localmicrobiological balance or significantly disrupt the protectiveglycoprotein vaginal coating. Disruption of the natural vaginal floraand/or removal or disruption of the protective glycoprotein vaginalcoating using conventional vaginal contraceptives can lead to irritationof the vaginal wall and/or lesions on the vaginal wall which can makethe transmission of STD easier and/or more likely. In addition, thecompounds useful in this invention are generally not disruptive torectal tissue and should not, therefore, significantly contribute to theformation of lesions or breaks in the rectal lining which could increasethe risk of STD transmission during anal intercourse. Moreover, theanti-microbial agents of the present invention, largely due to theirdual activities and site-specific delivery of nitric oxide, can be usedat lower concentrations, thereby reducing the risk of side effects orother adverse effects.

Either party to the sexual contact can employ the method of the presentinvention in order to protect him or herself and their partners. Thisfeature allows either party to take protective measures without relyingon the motivation or action of the other party. Of course, the highestlevel of protection is obtained when both or all parties takeappropriate steps to practice the methods of this invention inconjunction with “safe-sex” techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of percentage maximal acrosomal loss of NO7-SAMMA andNO23-SAMMA in the presence and absence of Ca²⁺ as well as theirrespective vector and NO-donor alone. The error bars represent 90%confidence limits. The bold horizontal lines in the chart area representthe predicted responses to NO7-SAMMA and NO23-SAMMA, respectively,assuming independence of the equivalent NO-donor and SAMMA responses inthe presence of Ca²⁺.

FIG. 2 is a comparison of SAMMA, NO23-SAMMA, and fractionated NO23-SAMMAas acrosomal loss stimuli.

FIG. 3 is a comparison of SAMMA and NO7-SAMMA for C. trachomatisinhibition. Elementary bodies were preincubated with agent (either SAMMAor NO7-SAMMA) for 4 hours at 0° C. before inoculation onto HeLa cells.

FIG. 4 is another comparison of SAMMA and NO7-SAMMA for C. trachomatisinhibition. HeLa cells were preincubated with agent (either SAMMA orNO7-SAMMA) for 4 hours at 37° C. before inoculation.

FIG. 5 is a comparison of fractionated NO23-SAMMA and nitrooxypropanolas acrosomal loss stimuli in the absence of Ca²⁺; thus, the activity offractionated NO23-SAMMA is due to the NO donor moiety.

FIG. 6 illustrates the inhibition of acrosomal loss induced byfractionated NO23-SAMMA by selective protein kinase G inhibitor KT5623.

FIG. 7 illustrates nitrite release from fractionated NO23-SAMMA in thepresence of 50 mM L-cysteine.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to improved anti-microbial agents andto methods for preventing STDs and/or reducing the risk of transmissionof such STDs through sexual activity using the improved anti-microbialagents. The anti-microbial agents comprises a delivery vector havinganti-microbial activity, and preferably contraceptive activity, coupledwith a nitric oxide donor moiety. The vector delivery component isdesigned to have affinity toward surface receptors required for targetcell recognition by spermatozoa (oocytes) and/or pathogenic microbes(susceptible tissue). It provides a vector-mediated targeted delivery ofNO, a naturally occurring, biologically active compound with knownactivities against spermatozoa and pathogenic microbes, including,though not restricted to, HIV, HSV, and C. trachomatis. NO is producedin situ by the NO donor covalently attached to the vector. Theanti-microbial agent is expected to bind with the targeted surfacereceptors on spermatozoa and/or pathogenic microbes; once bound,released NO can effect its known activities against the spermatozoaand/or pathogenic microbes in a very effective manner. Indeed, lowerconcentrations of the anti-microbial agent will be effective sincedelivery of the NO to the target organisms combined with the inherentactivity of the vector delivery component will be much more effective ascompared to either the vector component alone or NO donor alone.

The preferred contraceptive antimicrobial agents of this invention areespecially intended to prevent sexually transmitted infections andunplanned pregnancies. However, they may also have utility in preventingblood-borne pathogenic microbes from entering surrounding tissues.NO-coupled H₂SO₄-modified mandelic acids (NO-SAMMAs) are prototypes ofthis type of agent. They act by multiple mechanisms. They provide avector-mediated targeted delivery of NO, a naturally occurring,biologically active compound with known activities against spermatozoaand pathogenic microbes, including, though not restricted to, HIV, HSV,and C. trachomatis. NO is produced by a NO donor covalently attached tothe vector. The vector can be any one of several compounds with affinitytoward surface receptors required for target cell recognition byspermatozoa (oocytes) and pathogenic microbes (susceptible tissue).Examples of receptors on pathogenic microbes are collectively known asadhesins. Examples of receptors on spermatozoa have affinity towardoocyte-related proteins, such as the zone pellucida, and arecollectively known as heparin (or glycosaminoglycan) binding proteins orlectins. The vector is a ligand for these receptors.

Both vector and released NO contribute to its activity, each by one ormore mechanisms. The combination of these moieties on the same moleculeis more effective than either of the separate parts used alone, or incombination. This applies to the contraceptive and anti-microbialactivities of these agents, possibly by different mechanisms. The vector(ligand) promotes NO formation and biological activity in responsivecells, including spermatozoa, by a mechanism independent from that dueto the NO donor. The response is synergistic to the expected response tothe NO donor and ligand added in combination. The method of NO deliveryprovided by these new agents is more effective than would be provided bythe NO donor alone. The ligand portion of the molecule binds directly tothe spermatozoon or pathogenic microbe. NO released from the agent is indirect contact with the cell, allowing lower concentrations toaccomplish the same effect as the NO donor alone.

These new agents are expected to be more effective against pathogenicmicrobes than the ligand or NO donor used alone or in combination.Activity of NO-SAMMA against C. trachomatis supports this contention.The ligand is classified as an entry inhibitor. Entry inhibitors provideprotection against microbial invasion of susceptible cells, but probablyhave little beneficial effect against microbial survival or replication.NO-SAMMA and similar compounds increase the effectiveness of the ligandby providing a means of killing or otherwise inactivating the microbethrough the release of NO. Adhesin-like receptors have also beenidentified on potential target cells for microbial invasion. NO producedin these cells in response to interaction with NO-SAMMA and relatedcompounds could contribute to their anti-microbial activities.

Moreover, these new agents are expected to have broad anti-microbialactivity since the basic activity of the delivery vector component andthe released NO are present even in cases where the synergistic effectsnoted above may be absent. For example, not all microbes are sensitiveto NO. Thus, while NO-SAMMA does not offer enhanced activity against N.gonorrhoeae, the activity against this microbe due to SAMMA aloneremains. In other words, microbes do not have to be sensitive to both NOand the adhesin receptor antagonist to be affected by the anti-microbialcompounds of this invention. Of course, sensitivity to both NO and theadhesin receptor antagonist results in significantly increased kill orinhibition rates.

Preferred vector delivery components which are useful in the presentinvention for preparing anti-microbial agents are inhibitory agents suchas phosphorylated hesperidins, sulfonated hesperidins, polystyrenesulfonates, substituted benzenesulfonic acid formaldehyde co-polymers,H₂SO₄-modified mandelic acids, cellulose sulfates, and the like. Thus,preferred NO-coupled anti-microbial agents of this invention include,for example, phosphorylated hesperidins coupled with a NO-donor,sulfonated hesperidins coupled with a NO-donor, polystyrene sulfonatescoupled with a NO-donor, substituted benzenesulfonic acid formaldehydeco-polymers coupled with a NO-donor, H₂SO₄-modified mandelic acidscoupled with a NO-donor, cellulose sulfates coupled with a NO-donor, andthe like. Preferably the vector delivery components as well as theNO-coupled anti-microbial agents used in the present invention are atleast partially water soluble or water dispersable so that anti-STDformulations can more easily be prepared. Especially preferredanti-microbial agents for use in this invention include H₂SO₄-modifiedmandelic acids (SAMMAs) coupled with a NO-donor (i.e., NO-SAMMAs).

As noted above, preferred NO-SAMMAs include those which have beenfractionated so as to have a narrower molecular weight distribution andincreased activities (as measured against unfractionated material). Itis expected that the preparation of other NO-coupled anti-microbialagents having narrower molecular weight distributions will also haveincreased activities relative to their unfractionated counterparts. Suchnarrower molecular weight distributions can be obtained by fractionatingthe starting materials (e.g., SAMMA) or the final products (e.g.,NO-SAMMA) using conventional separation techniques; generally, it ispreferred that the starting materials be fractionated. Although notwishing to be limited by theory, it appears that the intermediatemolecular weight materials may have higher binding affinities forrelevant biological materials and thus higher activities. Of course, theoptimal molecular weight range for a given NO-coupled anti-microbialagent can be determined by routine experimentation. Moreover, theoptimal molecular weight range of a given NO-coupled antimicrobial agentmay vary depending on the activity measured (e.g., contraceptive,acrosomal loss, anti-HIV, anti-HSV, and/or the like activities), therelative amount of NO coupled to the agent, and the like.

SAMMA (i.e., H₂SO₄-modified mandelic acid), the most preferred vectordelivery component of this invention, is a carboxylated oligomer(average molecular weight of approximately 1.5 KDa) with contraceptiveand antimicrobial properties. Generally, SAMMA has a distribution ofcarboxylated oligomers having from 2-3 repeating units up to 20 or morerepeating units; typically the bulk of the material has 7-15 repeatingunits. Although not wishing to be limited by theory, it is thought thatthe reduction in the relative amounts of material having either low orhigh number of repeating unit by fractionation results in increasedactivities.

SAMMA is efficacious against HIV, HSV and C. trachomatis, among othersexually-transmitted pathogens. SAMMA is active against spermatozoa,inhibiting hyaluronidase and acrosin (two spermatozoal enzymes requiredfor fertilization), causes premature acrosomal loss, and iscontraceptive in the rabbit. (Zaneveld et at, “Use of mandelic acidcondensation polymer (SAMMA), a new antimicrobial contraceptive agent,for vaginal prophylaxis,” Fertil. Ster. 78: 1107-15 (2002); U.S. Pat.No. 5,925,621 (Jul. 20, 1999), U.S. Pat. No. 5,932,619 (Aug. 3, 1999),U.S. Pat. No. 6,028,115 (Feb. 22, 2000), and U.S. Pat. No. 6,239,182(May 29, 2001).) However, sperm motility is unaffected by SAMMA atconcentrations higher than those required for its antimicrobial andcontraceptive activities, suggesting that it is not acting by killingspermatozoa. Similar findings have been made regarding its antimicrobialproperties, insofar as minimal cytotoxic effects of SAMMA are seen onhost cells used for microbial infection in vitro. (Herold et at,“Mandelic acid condensation polymer novel candidate microbicide forprevention of human immunodeficiency virus and herpes simplex virusentry,” J. Virol. 76: 11236-44 (2002).)

Although research on its mechanisms of action is ongoing, SAMMA'santiviral effects are thought to be mediated, at least in part, by itsability to antagonize viral binding to target cells, mediated by theviral adhesins gp120 (for HIV) and g52 (for HSV). (Cheshenko et al.,“Candidate Topical Microbicides Bind Herpes Simplex Virus Glycoprotein Band Prevent Viral Entry and Cell-to-Cell Spread,” Antimicrob. AgentChemother. 48: 2025-36 (2004); Herold et at, “Mandelic acid condensationpolymer: novel candidate microbicide for prevention of humanimmunodeficiency virus and herpes simplex virus entry,” J. Virol. 76:11236-44 (2002).) SAMMA may be active against C. trachomatis by asimilar mechanism. Post-adhesion interference with viral-mediated signaltransduction at the level of the target cells remains to be determined.

SAMMA appears to induce premature acrosomal loss (AL) by aCa²⁺-dependent mechanism. Anderson et al., “SAMMA induces prematureacrosomal loss by Ca²⁺ signaling dysregulation,” J. Androl. 27: 568-577(2005). Although the initial point of interaction of SAMMA withspermatozoa (e.g., surface receptor(s)) is unknown, the process appearsto require entry of extracellular Ca²⁺. Unlike the physiologicalacrosome reaction, Ca²⁺ entry is likely mediated by voltage-dependentT-type Ca²⁺ channels (dose-dependent inhibition by diphenylhydantoin andNi²⁺), is unaffected by antagonism of InSP3 receptors, does not requirerelease of intracellular Ca²⁺ stores (not inhibited by 2-APB—an InSP3receptor antagonist and blocker of store-operated Ca²⁺ channels) and isnot mediated by protein kinase A (not inhibited by KT5720, a selectiveprotein kinase A inhibitor). SAMMA-induced acrosomal loss (SAL) requiresprotein kinase G and soluble guanylate cyclase (>95% inhibited by 2 μMKT5823—a selective protein kinase G inhibitor; ˜50% inhibited by 0.1 μMODQ—a selective inhibitor of soluble guanylate cyclase). Further, SAL isinhibited by inhibitors selective for the endothelial isoform of nitricoxide synthase. Taken together, these results suggest that SAL may bemediated by nitric oxide. Nitric oxide also likely mediates the acrosomereaction in response to physiological stimuli (e.g., progesterone andfollicular fluid; Herrero et al., “Evidence that nitric oxide synthaseis involved in progesterone-induced acrosomal exocytosis in mousespermatozoa,” Reprod. Fertil. Develop. 9: 433-9 (1997); Herrero et al.,“Progesterone enhances prostaglandin E2 production via interaction withnitric oxide in the mouse acrosome reaction,” Biochem. Biophys. Res.Commun.; 252: 324-8 (1998); Revelli et al., “Follicular fluid proteinsstimulate nitric oxide (NO) synthesis in human sperm: a possible rolefor NO in acrosomal reaction,” J. Cell Physiol. 178: 85-92 (1999);Herrero et al., “Nitric oxide interacts with the cAMP pathway tomodulate capacitation of human spermatozoa,” Free Rad. Biol. Med. 29:522-36 (2000)).

The second component of the NO-SAMMAs of the present invention is a NOdonor. NO donors alone also seem to induce AL, consistent with theproposed mechanism by which SAL occurs. However, unlike SAL, thesereactions do not appear to require Ca²⁺; the Ca²⁺ requirement for SAL islikely upstream from the action of NO. Nor do they appear to requireprotein kinase G (not inhibited by 2 μM KT5823 or 0.35 μMRp-8-Br-PET-cGMPS; Smolenski et al., “Functional analysis ofcGMP-dependent protein kinases I and as mediators of NO/cGMP effects,”Naunyn-Schmied Arch. Pharmacol., 358: 134-139 (1998)). These resultssuggest that the same outcome (AL) is produced by the same biologicallyactive intermediate (NO) produced by two stimuli/sources (SAMMA andNO-donors) by two independent mechanisms. Other work with NO-donorssuggest that AL in response to these agents may be mediated by acAMP-dependent mechanism. (Kurjak et al., “NO releases bombesin-likeimmunoreactivity from enteric synaptosomes by cross-activation ofprotein kinase A,” Amer. J. Physiol. 276: G1521-G30 (1999); Vila-Petroffet al., “Activation of distinct cAMP-dependent and cGMP-dependentpathways by nitric oxide in cardiac myocytes,” Circul. Res. 84: 1020-31(1999).)

NO is a highly bioactive, short-lived gaseous molecule produced inresponse to various physiological stimuli. Among its actions are smoothmuscle relaxation, inhibition of platelet aggregation and adhesion,neurotransmission, regulation of apoptosis, and cytotoxicity. Further,NO is toxic to a number of bacteria, viruses, and other foreignparticles. (Gross et al., “Nitric oxide: pathophysiological mechanisms,”Ann. Rev. Physiol. 57: 737-69 (1995).) Specifically, NO appear to play akey role in the natural defense against microbes, including HIV and HSV,and Chlamydia.

Viral enzymes (e.g., proteases, reverse transcriptases, ribonucleotidereductase) containing cysteine residues are targets for NO-mediatednitrosylation; viral-encoded transcription factors are also targets.(Persichini et al., “Cysteine nitrosylation inactivates the HIV-1protease,” Biochem. Biophys. Res Commun. 250: 575-6 (1998); Broillet,“S-nitrosylation of proteins,” Cell Mol. Life Sci. 55: 1036-42 (1999);Persichini et al., “Molecular bases for the anti-HIV-1 effect of NO.Commentary,” int. J. Mol. Med. 4: 365-8 (1999); Benz et al., “Tonalnitric oxide and health: antibacterial and viral actions andimplications for HIV,” Med. Sci Monitor. 8: RA27-RA31 (2002).) NOappears to react with and disrupt structural proteins essential forviral replication. (Saavedra et al., “The secondary amine/nitric oxidecomplex ion R₂N[N(O)NO]⁻ as nucleophile and leaving group in S(N)Arreactions,” J. Org. Chem. 66: 3090-8 (2004) Stimulus-induced NOproduction is beneficial in preventing HIV infection, and the protectiveeffect of low levels of NO on cell survival in the CNS subsequent to HIVinfection is recognized. (Fiscus, “Involvement of cyclic GMP and proteinkinase G in the regulation of apoptosis and survival in neural cells,”Neurosig. 11: 175-90 (2002).)

Endogenous or exogenous NO inhibits HSV-1 and HSV-2 infectivity. Agentslowering or antagonizing NO exacerbate HSV infection. Inducible nitricoxide synthase (INOS) inhibition increases pathology and viral titers ofHSV-2-infected mice. (Benencia et al., “Effect of aminoguanidine, anitric oxide synthase inhibitor, on ocular infection with herpes simplexvirus in Balb/c mice,” Invest. Ophthalmol. Vis. Sci. 42: 1277-84 (2001);Benencia et al., “Nitric oxide and HSV vaginal infection in BALB/cmice,” Virology 309: 75-84 (2003). Macrophage iNOS activation forms partof the innate immune response to HSV. (Croen, “Evidence for antiviraleffect of nitric oxide. Inhibition of herpes simplex virus type 1replication,” J. Clin. Invest. 91: 2446-52 (1993); Benencia et al.,“Nitric oxide and macrophage antiviral extrinsic activity,” Immunology98: 363-70 (1999); Paludan et al., “Interferon (IFN)-gamma and Herpessimplex virus/tumor necrosis factor-alpha synergistically induce nitricoxide synthase 2 in macrophages through cooperative action of nuclearfactor-kappa B and IFN regulatory factor-1,” Eur. Cytokine Netw. 12:297-308 (2001).) Macrophage NO inhibits HSV-1 replication, and iNOSinhibition increases HSV-1 titers. (Karupiah et al., “Inhibition ofviral replication by nitric oxide and its reversal by ferrous sulfateand tricarboxylic acid cycle metabolites,” J. Exp. Med. 181: 2171-9(1995); Kodukula et al., “Macrophage control of herpes simplex virustype 1 replication in the peripheral nervous system,” J. Immunol. 162:2895-905 (1999).) iNOS-deficient knockout mice are more susceptible toHSV-1. (MacLean et al, “Mice lacking inducible nitric-oxide synthase aremore susceptible to herpes simplex virus infection despite enhanced Th1cell responses,” J. Gen. Virol. 79: 825-30 (1998).) NOS inhibitorsincrease HSV-1-induced pathology. (Benencia et al., “Nitric oxide andmacrophage antiviral extrinsic activity,” Immunology 98: 363-70 (1999).)HSV elimination from the CNS requires NO. (Chesler et at, “The role ofIFN-gamma in immune responses to viral infections of the central nervoussystem,” Cytokine Grth. Fact. Rev. 13: 441-54 (2002).) iNOS-derived NOinhibits viral replication, (Benencia et at, “Effect of aminoguanidine,a nitric oxide synthase inhibitor, on ocular infection with herpessimplex virus in Balb/c mice,” Invest. Ophthelmol. Vis. Sci. 42: 1277-84(2001); Kodukula et al., “Macrophage control of herpes simplex virustype 1 replication in the peripheral nervous system,” J. Immunol. 162:2895-905 (1999); Adler et al. “Suppression of herpes simplex virus type1 (HSV-1)-induced pneumonia in mice by inhibition of inducible nitricoxide synthase (iNOS, NOS2),” J. Exp. Med. 185: 1533-40 (1997); Fuji′ etal., “Role of nitric oxide in pathogenesis of herpes simplex virusencephalitis in rats,” Virology 256: 203-12 (1999).)

NO inhibits C. trachomatis growth. (Igietseme et at, “Inhibition ofintracellular multiplication of human strains of Chlamydia trachomatisby nitric oxide,” Biochem. Biophys. Res. Commun, 232: 595-601 (1997).)NO production by epithelial and possibly T-cells is important for theresolution of chlamydial infections. IFN-γ prevents C. trachomatisreplication and promotes NO formation; both are inhibited by iNOSinhibitors. (Mayer et al., “Gamma interferon-induced nitric oxideproduction reduces Chlamydia trachomatis infectivity in McCoy cells,”Infect. Immun. 61: 491-7 (1993); Devitt et al., “Induction of alpha/betainterferon and dependent nitric oxide synthesis during Chlamydiatrachomatis infection of McCoy cells in the absence of exogenouscytokine,” infect Immun 64: 3951-6 (1996).) NO donors inhibit C.trachamatis replication in epithelial cells. (Igietseme et al.,“inhibition of intracellular multiplication of human strains ofChlamydia trachomatis by nitric oxide,” Biochem. Biophys. Res. Commun.232: 595-601 (1997).) Protection against chlamydial infection by T-cellscorrelates with their ability to induce NO production. (Igietseme, “Themolecular mechanism of T-cell control of Chlamydia in mice: role ofnitric oxide,” Immunology 87: 1-8 (1996).) NOS inhibition increasesbacterial titers of infected mice and impairs the ability of T-cellclones to clear genital chlamydial infection. (Igietseme, “Molecularmechanism of T-cell control of Chlamydia in mice: role of nitric oxidein vivo,” Immunology 88: 1-5 (1996).) This is also seen in macrophages,with a strong correlation between NOS activity and chlamydialinhibition, effects antagonized by NOS inhibition. (Chen et al., “Nitricoxide production: a mechanism of Chlamydia trachomatis inhibition ininterferon-gamma-treated RAW264.7 cells,” FEMS Immunol. Med. Microbiol.14: 109-20 (1996); Azenabor et al., “Chlamydia pneumoniae survival inmacrophages is regulated by free Ca²⁺ dependent reactive nitrogen andoxygen species,” J. Infect. 46: 120-8 (2003).) Mouse strains thatproduce more NO are more resistant to C. trachomatis. (Ramsey et al.,“Role for inducible nitric oxide synthase in protection from chronicChlamydia trachomatis urogenital disease in mice and its regulation byoxygen free radicals,” Inf. Immun. 69: 7374-9 (2001).) Macrophages fromchlamydia-infected, IFN-γ receptor-deficient mice have increasedchlamydial titers, and no detectable NO. (Johansson et al., “Genitaltract infection with Chlamydia trachomatis fails to induce protectiveimmunity in gamma interferon receptor-deficient mice despite a stronglocal immunoglobulin A response,” Inf. Immun. 65: 1032-44 (1997).) IniNOS-deficient mice, IFN-γ is bacteriostatic against chlamydialinfection. However, IFN-γ is bactericidal in iNOS-sufficient mice anderadicates the microbe. (Ramsey et al., “Chlamydia trachomatispersistence in the female mouse genital tract: inducible nitric oxidesynthase and infection outcome,” Infect. Immun. 69: 5131-7 (2001).)

The present invention, in an especially preferred form, combines SAMMAand a NO donor in a single compound or molecule which provides thebenefits of both components in a synergistic manner. As noted above, (1)SAL occurs by a Ca²⁺- and NO-dependent mechanism; (2) NO release from NOdonors induces AL by a Ca²⁺-independent mechanism, distinct from thatresponsible for SAL; and (3) NO has antiviral and antibacterialactivities, and has a key role in the natural defense against HIV, HSVand C. trachomatis infections. It was hoped that both the contraceptiveand antimicrobial activities of SAMMA could be improved through thecovalent attachment of an NO donor. This has proven to be the case;indeed the degree of improvement has been surprising.

The NO-SAMMA compound of the present invention was found to have thefollowing properties: (1) induces AL in the presence or absence of Ca²⁺;(2) AL due to NO release (in the absence of Ca²⁺) is effected by a lowerconcentration of NO-donor equivalents present in the derivative thanrequired for NO-donor alone (not wishing to be limited by theory, thiseffect is thought to be largely due to the fact that the source of NOwould be directed to the surface of the target cell); (3) activity ofthe derivative against spermatozoa is synergistic compared with eitherNO-donor or SAMMA alone (again, not wishing to be limited by theory,this effect is thought to be due to different mechanisms by which theyinduce AL); (4) antimicrobial activity due to NO release occurs at lowerconcentration of NO-donor equivalents present in the derivative thanrequired for NO donor alone; and (5) a synergistic antimicrobial effectcompared to either NO-donor or SAMMA alone has been found. It appearsthat the SAMMA moiety inhibits microbial binding to target or host cellsand the NO produced by the NO-donor moiety kills or otherwise disruptsthe invasive cells (i.e., microbes and/or spermatozoa).

The NO-donor moieties suitable for use in the present invention must becapable of being attached, preferably covalently, to the vector deliverycomponent and of releasing NO during use. Suitable NO-donor moieties canbe derived from nitrate esters, furoxans, ketoximes, S-nitrosothiols,nitrosohydrazines, hydroxylamides, and the like. Of course, otherNO-donor moieties can be used if desired so long as they meet theconditions required for the present invention.

Nitrate esters may release NO by several routes, such as, for example,

RONO₂+2e ⁻+H⁺→ROH+NO₂ ⁻

NO₂ ⁻ +e ⁻+H⁺→HO⁻+NO

NO₂ ⁻+M^(n+)→M^((n+1))+.O+NO

where R is an alkyl group preferably having 2 to 8 carbon atoms, andmore preferably 2 to 6 carbon atoms, and M′ is a metal ion (e.g., Fe⁺,Cu⁺, Cr²⁺, Co²⁺, and the like. NO may also be released from such nitrateesters by a thiol-activated scheme:

Furoxans may also release NO via a thiol-activated scheme, asillustrated below (using 1,2,5-oxiadiazole-2-oxide as an example):

Conjugated ketoximes, using (±)-E-4-ethyl-2-[(E)-hydroxyimino]-5nitro-3-hexenamide (NOR-3) as an example, can release NO via thefollowing scheme:

NOR-3 (0.5 mM in 0.1 M PBS) has a half-life of about 30 minutes at pH7.4 and 37° C. Likewise, imines can also be used as NO-donor moieties.For example, 3-(4-morpholinyl)sydnomine (SIN-1) can release NO throughthe following reaction scheme:

S-nitrosothiols, as shown below for S-nitroso-N-acetylpenicillamine(SNAP) and S-nitrosoglutathione, can also produce NO:

NO can also be released from 2-nitroso hydrazine derivatives, includingdiazeniumdiolates, such as1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5) and-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7),as indicated below:

(Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino}-diazen-1-ium-1,2-diolate](shown below; spermine-NONOate) is thought to release NO by a similarmechanism:

Hydroxylamides can also be used as the NO-donor moieties. For example,hydroxyurea reacts with hemoglobin to produce iron nitrosyl hemoglobin,nitrite, and nitrate, thereby releasing NO. Hydroxyurea can also releaseNO via the peroxidase-mediated hydrolysis of hydroxyurea tohydroxylamine.

For contraceptive antimicrobial activity, the vector can be any compoundthat blocks spermatozoal binding to the oocyte or is otherwisecontraceptive (a surrogate marker for this activity is the ability ofthe vector to induce premature acrosomal loss in vitro) and blocksmicrobial binding to the target (host) cells. By itself, it should havecontraceptive and antimicrobial activities. Specifically, the vectorshould be an adhesin antagonist (receptor analog). General examplesinclude polyanionic polymers and/or oligomers with affinity for adhesinsthat bind to heparan sulfate or other glycosaminoglycans. Exampleswithin this class include cellulose sulfate, polystyrene sulfonate,dextran sulfate, naphthalenesulfonic acid polymer (e.g., Pro2000;Indevus Pharmaceuticals, Lexington, Miss.), polymethylene hydroquinonesulfonic acid, or sulfuric acid modified mandelic acid (SAMMA). Theseagents have activities against HIV-1, HSV-1, HSV-2, Chlamydiatrachomatis, and Neisseria gonorrhoeae. Based on adhesin receptorspecificities, other pathogens that should be affected by these agentsand NO, include Streptococcus spp (see, e.g., Puliti et al., “inhibitionof nitric oxide synthase exacerbates group B streptococcus sepsis andarthritis in mice,” Inf. Immun. 72: 4891-4 (2004); Kerr et al., “Nitricoxide exerts distinct effects in local and systemic infections withStreptococcus pneumoniae,” Microb. Pathogen. 36: 303-10 (2004); Ozturket al., “Serum and mucosal nitric oxide levels and efficacy of sodiumnitroprusside in experimentally induced acute sinusitis,” Yonsei. Med.J. 44: 424-8 (2003); Leib et al., “Inducible nitric oxide synthase andthe effect of aminoguanidine in experimental neonatal meningitis,” J.Inf. Dis. 177: 692-700 (1998)), Staphylococcus spp (Nablo et al.,“Nitric oxide-releasing sol-gels as antibacterial coatings fororthopedic implants,” Biomaterials 26: 917-24 (2005); Zhang et al.,“Differential antibacterial activity of nitric oxide from theimmunological isozyme of nitric oxide synthase transduced intoendothelial cells,” Nitric Oxide 7: 42-9 (2002)), Mycoplasma spp(Hickman-Davis et al., “Cyclophosphamide decreases nitrotyrosineformation and inhibits nitric oxide production by alveolar macrophagesin mycoplasmosis,” Inf. Immun. 69: 6401-10 (2001); Hickman-Davis et al.,“Surfactant protein A mediates mycoplasmacidal activity of alveolarmacrophages by production of peroxynitrite,” Proc. Nat. Acad. Sci. USA96: 4953-8 (1999)), Mycobacterium spp (Smeulders et al.,“S-Nitrosoglutathione cytotoxicity to Mycobacterium smegmatis and itsuse to isolate stationary phase survival mutants,” FEMS Microbiol. Lett.239: 221-8 (2004)), Mycoplasma spp (Bogdan, “Reactive oxygen andreactive nitrogen metabolites as effector molecules against infectiouspathogens,” in The innate immune response to infection (Kaufmann et al.,eds.), Washington, D.C.: ASM Press, p. 357-96 (2004)), Listeriamonocytogenes (Carryn et al, “Impairment of growth of Listeriamonocytogenes in THP-1 macrophages by granulocyte macrophagecolony-stimulating factor: release of tumor necrosis factor-alpha andnitric oxide,” J. Inf. Dis. 189: 2101-9 (2004); Myers et al., “Localizedreactive oxygen and nitrogen intermediates inhibit escape of Listeriamonocytogenes from vacuoles in activated macrophages,” J. Immunol. 171:5447-53 (2003); Remer et al., “Nitric oxide is protective in listericmeningoencephalitis of rats,” Inf. Immun. 69: 4086-93 (2001)),Helicobacter pylori (Bussiere et al., “Spermine causes loss of innateimmune response to Helicobacter pylori by inhibition of induciblenitric-oxide synthase translation,” J. Biol. Chem. 280: 2409-12. (2005):Potter et al., “Exogenous nitric oxide inhibits apoptosis in guinea piggastric mucous cells,” Gut 46: 156-62 2000), Borrelia spp (Lusitani etal., “Borrelia burgdorferi are susceptible to killing by a variety ofhuman polymorphonuclear leukocyte components,” J. Infect. Dis. 185:797-804 (2002)), and Bordella pertussis (Canthaboo et al.,“Investigation of role of nitric oxide in protection from Bordetellapertussis respiratory challenge,” Infect. Immun. 70: 679-84 (2002);Torre et al., “Regulation of inflammatory responses to Bordetellapertussis by N(G)-monomethyl-L-arginine in mice intranasally infected,”Mediat. Inflam. 8: 25-9 (1999)). Table 1 below provides a summary ofexamples of contraceptive antimicrobial vector/NO donor combinations foruse in the present invention.

For non-contraceptive antimicrobial activity, the vector shouldantagonize microbial adhesin binding to target cells. The targetedmicrobe should be sensitive to NO. For example, fibronectin antagonists(Ofek et al., “Adhesins, receptors, and target substrata involved in theadhesion of pathogenic bacteria to host cells and tissues,” in Bacterialadhesion to animal cells and tissues, Washington, D.C.; ASM Press, p.177-405 (2003)) and NO are effective against Borrelia spp (Lusitani etal., “Borrelia burgdorferi are susceptible to killing by a variety ofhuman polymorphonuclear leukocyte components,” J. Infect. Dis. 185:797-804 (2002)), Chlamydia trachomatis (Chen et al., “Nitric oxideproduction: a mechanism of Chlamydia trachomatis inhibition ininterferon-gamma-treated RAW264.7 cells,” FEMS Immunol. Med. Microbiol.14: 109-20 (1996); Azenabor et al., “Chlamydia pneumoniae survival inmacrophages is regulated by free Ca²⁺ dependent reactive nitrogen andoxygen species,” J. Infect. 46: 120-8 (2003); Igietseme “Molecularmechanism of T-cell control of Chlamydia in mice: role of nitric oxidein vivo,” immunology 88: 1-5 (1996); Igietseme et al., “Inhibition ofintracellular multiplication of human strains of Chlamydia trachomatisby nitric oxide,” Biochem. Biophys. Res. Commun. 232: 595-601 (1997)),Streptococcus spp (Puliti et al., “Inhibition of nitric oxide synthaseexacerbates group B streptococcus sepsis and arthritis in mice,” Infect.Immun. 72: 4891-4 (2004); Kerr et al., “Nitric oxide exerts distincteffects in local and systemic infections with Streptococcus pneumoniae,”Microb. Pathogen. 36: 303-10 (2004)), Fusobacterium nucleatum (Allakeret al., “Antimicrobial effect of acidified nitrite on periodontalbacteria,” Oral Microbiol. Immunol. 16: 253-6 (2001)), Mycobacterium spp(Bogdan, “Reactive oxygen and reactive nitrogen metabolites as effectormolecules against infectious pathogens,” in The innate immune responseto infection (Kaufmann et al., eds.), Washington, D.C.: ASM Press, p.357-96 (2004); Yamashiro et al., “Lower expression of Th1-relatedcytokines and inducible nitric oxide synthase in mice withstreptozotocin-induced diabetes mellitus infected with Mycobacteriumtuberculosis,” Clin. Exp. Immunol. 139: 57-64 (2005); Copenhaver et al.,“A mutant of Mycobacterium tuberculosis H37Rv that lacks expression ofantigen 85A is attenuated in mice but retains vaccinogenic potential,”Infect. Immun. 72: 7084-95 2004)), Porphyromonas gingivalis (Bogdan,“Reactive oxygen and reactive nitrogen metabolites as effector moleculesagainst infectious pathogens,” in The Innate immune response toinfection (Kaufmann et al., eds.), Washington, D.C.: ASM Press, p.357-96 (2004)), Salmonella enterica (Bogdan, “Reactive oxygen andreactive nitrogen metabolites as effector molecules against infectiouspathogens,” in The innate immune response to infection (Kaufmann et al.,eds.), Washington, D.C.: ASM Press, p. 357-96 (2004)), Staphylococcusspp (Nablo et al., “Nitric oxide-releasing sot-gels as antibacterialcoatings for orthopedic implants,” Biomaterials 26: 917-24 (2005); Zhanget al., “Differential antibacterial activity of nitric oxide from theimmunological isozyme of nitric oxide synthase transduced intoendothelial cells,” Nitric Oxide 7: 42-9 (2002)), and Yersinia spp(Dykhuizen et al., “Antimicrobial effect of acidified nitrite on gutpathogens: importance of dietary nitrate in host defense,” Antimicrob.Agents Chemother. 40: 1422-5 (1996); Campos-Perez et al., “Toxicity ofnitric oxide and peroxynitrite to bacterial pathogens of fish,” Dis.Aquat. Org. 43: 109-15 (2000)), as welt as the parasites Trichomonasvaginalis (Crouch et al., “Binding of fibronectin by Trichomonasvaginalis is influenced by iron and calcium,” Microb. Pathogen. 31:131-44 (2001); Gradoni et al., “Nitric′ oxide and anti-protozoanchemotherapy,” Parassitologia. 46: 101-3 (2004)) and Leishmania spp(Bogdan, “Reactive oxygen and reactive nitrogen metabolites as effectormolecules against infectious pathogens,” in The innate immune responseto infection (Kaufmann et al., eds.), Washington, D.C.: ASM Press, p.357-96 (2004)). Table 2 below provides a summary of examples ofnon-contraceptive antimicrobial vector/NO donor combinations suitablefor use in the present invention.

Other suitable adhesin receptor antagonists include, for example,lactosyl- and galactosylceramides, laminin fragments, peptidoglycans,and glycopeptides. (See, e.g., Ofek et al., “Adhesins, receptors, andtarget substrata involved in the adhesion of pathogenic bacteria to hostcells and tissues,” in Bacterial adhesion to animal cells and tissues,Washington, D.C.: ASM Press, p. 177-405 (2004)

TABLE 1 Contraceptive antimicrobial vectors for enhancement by nitricoxide: target microbes Adhesin/adhesion molecule specificity VectorMicrobe Nitric oxide effect heparan sulfate, cellulose sulfate; HIV NOdonors inhibit HIV-1 reverse transcriptase; protective effect of NO onheparan and other polystyrene CNS after HIV infection sulfatedsulfonate; HSV NOS inhibitors increase viral titers in mice; NO inhibitsviral replication glycosaminoglycans; dextran sulfate; Chlamydia spp NOdonors inhibit C. trachomatis replication; NOS inhibitors increasebacterial sulfated sugars; SAMMA; titers in mice sulfated polymethylene-Bordetella pertussis NO (iNOS) protects mice against Bordetellapertussis infection and decreases polysaccharides; hydroquinonemortality to Bordetella pertussis infection in vivo glycosaminoglycans;sulfonate Borrelia spp NO kills Borrelia in vitro hyaluronan;sulfomucin; Helicobacter pylori NO contributes to killing H. pylori inmacrophages and protects gastric cells sulfated glycoprotein; from H.pylori-induced apoptosis sulfated glycolipids; Listeria NO contributesto impairment of intracellular growth of L. monocytogenes, sulfatedmonocytogenes retention of L. monocytogenes by activated macrophages andprotects against glycoconjugates; listeric meningoencephalitis in ratssulfated proteoglycans Mycobacterium spp Bactericidal action of NO donoragainst M. smegmatis Mycoplasma spp iNOS-deficient mice have highertiters than controls after infection with M. pulmonis Streptococcus sppmortality of mice infected with Group B streptococcus increased by NOSinhibitor; protective effect of NO against GBS-induced meningitis inrats; direct inhibition of S. pneumoniae by NO and NO donorStaphylococcus spp NO donors and NOS-derived NO kill Staph and protectagainst Staph infections Plasmodium control of infection in human tissueby NO-mediated mechanisms falciparum (protozoon) Trypanosoma sppintracellular and extracellular morphotypes of Trypanosoma killed by NOin (protozoon) vitro and in vivo Leishmania spp NO produced bymacrophages kills intracellular Leishmania (pratozoon) Giardia(pratozoon) parasitostatic effect of NO donors

TABLE 2 Noncontraceptive antimicrobial vectors for enhancement by nitricoxide: target microbes Adhesin/adhesion molecule specificity VectorMicrobe Nitric oxide effect Fibronectin Fibronectin Borrelia spp NOinhibits Borrella fragments (e.g., Chlamydia inhibition by macrophagesantagonized end bacterial titer in mice peptides 4-15 trachomatisincreased by NOS inhibitors; replicaton inhibited by NO donors residuesin Streptococcus spp NOS inhibitors increase mortality of infected mice;inhibition by length that NO donors contain the Fusobacteriumbactericidal effect of nitrite (potential NO precursor) recognitionnucleatum sequence Mycobacterium spp NO contributes to pathogen control;reduced NO production in Arg-Gly-Asp or diabetics impairs defenseagainst M. tuberculosis; inhibited by Arg-X-Asp-Ser NO donors orLeu-Ile-Gly- Porphyromonas NO contributes to pathogen centralArg-Lys-Lys) gingivalis Salmonella enterica NO is essential for pathogencontrol Yersinia spp bactericidal effects of acidified nitrite;inhibited by NO donors Leishmania NO is essential for pathogen control(protozoon) Naegleria fowleri NO and/or NO donors kill Naegleria

Although not wishing to be limited by theory, it appears thatNO-couplings to produce the agents in the present invention will alsoimprove the safety profile of vector units such as SAMMA, sulfonatedhesperidins, phosphorylated hesperidins, polystyrene sulfonate,cellulose sulfate and the like, intended for use to prevent HIVinfection. This is thought to be due primarily to the fact that NOdonors inhibit production and actions of pro-inflammatory cytokines.

Concerns have been expressed regarding the ability of microbicides toincrease production of inflammatory cytokines, which can causeproliferation of target cells for HIV infection (Keller et al.,“Rigorous pre-clinical evaluation of topical microbicides to preventtransmission of human immunodeficiency virus,” J. Antimicrob. Chemother.51: 1099-1102 (2003); Keller et al., “Topical microblcides for theprevention of genital herpes infection,” J. Antimicrob. Chemother. 55:420-423 (2005); Stone, “Microbicides: a new approach to preventing HIVand other sexually transmitted infections,” Nature Reviews 1: 977-85(2002)). NO donors as provided by the present invention are thought tobe beneficial in this context. NO donors either reduce production ofinflammatory cytokines or reduce their actions. The extent of theseactions is at least partially dependent upon the cell type and level ofNO (Proud, “Nitric oxide and the common cold,” Cur. Opin. Allergy Clin.,Immunol. 5: 37-42 (2005)).

Vaginal application of the NO donor isosorbide mononitrate to women hasno effect on the production of interleukin (IL)-1, IL-8, IL-8, IL-10,IL-15, tumor necrosis factor (TNF)-α, or monocyte chemoattractantprotein-1 (Ledingham et al., “Nitric oxide donors stimulateprostaglandin F(2alpha) and inhibit thromboxane B(2) production in thehuman cervix during the first trimester of pregnancy,” Mel. Hum. Reprod.5: 973-982 (1999)). On the other hand, molsidomine, a precursor to theNO donor SIN-1, decreases levels of the pro-inflammatory cytokinesTNF-α, IL-1beta and IFN-gamma and increases production of theanti-inflammatory cytokines IL-6 and IL-10 in ischemic renal cells(Rodriguez-Pena et al., “Intrarenal administration of molsidomine, amolecule releasing nitric oxide, reduces renal ischemia-reperfusioninjury in rats,” Amer. J. Transplant. 4: 1605-1613 (2004)). Similarbeneficial effects of this NO donor have been seen in rats withexperimental allergic encephalomyelitis (Kwak et al., “Molsidomineameliorates experimental allergic encephalomyelitis in Lewis rats,”Immunopharmacol. Immunotoxicol. 25: 41-52 (2003)).

The NO donor SNAP decreases production of the pro-inflammatory cytokine,IL-12, in mouse macrophages (Xiong et al., “Inhibition of interleukin-12p40 transcription and NF-kappaB activation by nitric oxide in murinemacrophages and dendritic cells,” J. Biol. Chem. 279: 10776-10783(2004)). Similar inhibitory activity is seen in human peripheral bloodmononuclear cells (Rachlis et al., “Nitric oxide reduces bacterialsuperantigen-immune cell activation and consequent epithelialabnormalities,” J. Leukocyte Biol. 72: 339-346 (2002)), in activatedhuman pulmonary microvascular endothelial cells (Jiang et al., “Effectsof antioxidants and NO on TNF-alpha-induced adhesion molecule expressionin human pulmonary microvascular endothelial cells,” Resp. Med. 99:580-91 (2005)), rat basophilic leukemia cells (Heywood et al.,“Nicorandil inhibits degranulation and TNF-alpha release from RBL-2H3cells,” Inflam. Res. 51: 176-181 (2002)), colonic tissue from colitic(induced colitis) mice (Sales et al., “Nitric oxide supplementationameliorates dextran sulfate sodium-induced colitis in mice,” Lab.Invest. 82: 597-607 (2002)) and in lipopolysaccharide (LPS)-inducedairway inflammation in mice (Lagente et al., “A nitric oxide-releasingsalbutamol elicits potent relaxant and anti-inflammatory activities,” J.Pharmacol. Exp. Ther. 310: 357-375 (2004).

The NO donor NOC-18 reduces the number of IFN-gamma-secreting CD4+ Tcells in patients with unstable angina and coronary spastic angina(Soejima et al., “Preference toward a T-helper type 1 response inpatients with coronary spastic angina,” Circulation 107: 2196-2200(2003)), and the NO donor nitroprusside decreases production ofpro-inflammatory cytokines after reperfusion in coronary artery bypassgraft (Freyholdt et al., “Beneficial effect of sodium nitroprussideafter coronary artery bypass surgery: pump function correlates inverselywith cardiac release of proinflammatory cytokines,” J. Cardiovasc.Pharmacol. 42: 372-378 (2003)). NO donors inhibit stimulus-inducedincreases in pro-inflammatory cytokines, while having little effect onresting levels.

NO produced in response to IL-1β and TNF-α stimulation causes increasedcyclooxygenase 2 (COX-2) expression in mesangial cells. However, NOdonors decrease COX-2 in these cells, likely through feedback inhibition(Diaz-Cazorìa et al., “Dual effect of nitric oxide donors oncyclooxygenase-2 expression in human mesangial cells,” J. Amer. Soc.Nephrol. 10: 943-952 (1999)). NO donors exert feedback inhibition on NOproduction in response to inflammatory cytokines and LPS (Galley et al.,“Regulation of nitric oxide synthase activity in cultured humanendothelial cells: effect of antioxidants,” Free Rad. Biol. Med. 21:97-101 (1996)). IL-1beta-stimulated chondrocytes show increasedactivation of NFkappaB, an effect that is abrogated by the NO donor Snitrosocysteine ethyl ester (Clancy et al., “Nitric oxide sustainsnuclear factor kappaB activation in cytokine-stimulated chondrocytes,”Osteoarthrit. Cartil. 12: 552-558 (2004)). Similar effects are noted inhuman mesangial cells (Diaz-Cazorla et al., “Dual effect of nitric oxidedonors on cyclooxygenase-2 expression in human mesangial cells,” J.Amer. Soc. Nephrol. 10: 943-952 (1999)) and in mouse macrophages (Xionget al., “Inhibition of interleukin-12 p40 transcription and NF-kappaBactivation by nitric oxide in murine macrophages and dendritic cells,”J. Biol. Chem. 279: 10775-10783 (2004)). Pretreatment of human mesangialcells with the NO donors SIN-1 or nitroprusside decreases the subsequentincrease in NFkappaB binding and macrophage chemoattractant protein-1(MCP-1) expression in response to TNF-α or IL-1β (Lee et al., “Exogenousnitric oxide inhibits tumor necrosis factor-alpha- orinterleukin-1-beta-induced monocyte chemoattractant protein-1 expressionin human mesangial cells. Role of IkappaB-alpha and cyclic GIMP,”Nephron 92: 780-787 (2002)). Increased permeability of microvesselsmaking up the blood-brain barrier in response to IL-15, IFN-γ, and LPSis reversed by NO donors (Wong et al., “Cytokines, nitric oxide, andcGMP modulate the permeability of an in vitro model of the humanblood-brain barrier,” Exp. Neurol. 190: 446-455 (2004)). Further, NOdonors promote apoptosis of activated macrophages (Niinobu et al.,“Negative feedback regulation of activated macrophages via Fas-mediatedapoptosis,” Amer. J. Physiol. 279: 0504-0509 (2000)), which are targetsfor HIV infection. This effect may depend on the NO donor concentration(and by inference, the NO concentration) used (von Knethen et al.,“NF-kappaB and AP-1 activation by nitric oxide attenuated apoptotic celldeath in RAW 264.7 macrophages,” Mol. Biol. Cell 10: 361-372 (1999)).Proliferation of T- and other immune cells and their recruitment inresponse to TNF-α, IL-2 or LPS is substantially reduced by several NOdonors (Corinti et al., “Regulatory role of nitric oxide onmonocyte-derived dendritic cell functions,” J. Interfer. Cytokine Res.23: 423-431 (2003); Haider et al., “Dual functionality ofcyclooxygenase-2 as a regulator of tumor necrosis factor-mediated G1shortening and nitric oxide-mediated inhibition of vascular smoothmuscle cell proliferation,” Circulation 108: 1015-1021 (2003): Macphailet al., “Nitric oxide regulation of human peripheral blood mononuclearcells: critical time dependence and selectivity for cytokine versuschemokine expression,” J. Immunol. 171: 4809-4815 (2003)).

The above findings suggest that incorporation of an NO donor into atopical microbicide as provided by this invention may, among othereffects, improve its safety profile. NO donors inhibit the productionand actions of inflammatory cytokines that may act to proliferate targetcells for HIV infection.

The synthesis of the NO donor/vector adduct of the adhesin receptorantagonist can be achieved by standard organic synthetic pathways, inwhich the NO donor moiety is attached to a spacer molecule (e.g., alkaneof 2-8 carbons in length) that contains a moiety suitable for couplingto the vector (e.g., amino, carboxyl, or hydroxyl). The spacer moleculecontaining the NO donor can be linked to hydroxyl, amino or carboxylmoieties of the vector via an ester or amide linkage. In instances wherethe vector contains polyol groupings (e.g., dextran or cellulosederivatives), regioselective attachment of the NO donor as a nitrateester can be effected. Representative examples of such synthetic methodsare presented below; of course, other methods can be used to prepare theNO donor/vector adducts of this invention.

Nitrate Esters of SAMMA.

Reaction of the bromoalkanols in anhydrous acetonitrile with silvernitrate affords the required nitrooxy-alkanols as shown in Equation 1for bromalkanols ranging in size from C2 to C6.

Of course, values of n greater than 5 can be used if desired for thenitrate esters in Equations 1-4. As shown in Equation 2,substoichiometric coupling to SAMMA free acid is accomplished with1,1′-carbonyldiimidazole (CDI) in dry DMF linking the nitrooxy alkanol(1,3-nitrooxyphenol) by an ester linkage. (Endres et al., “NO-donors,part 3: nitrooxyacylated thiosalicylates and salicylates—synthesis andbiological activities,” Eur. J. Med, Chem. 34: 895-901 (1999).)

A series of nitrooxyalkyl amines C2-C8 can be prepared, that can becoupled to SAMMA by an amide linkage. Focus is placed on the C3 adductat levels of substitution from 5-60%. Minin and Walton (“Radical RingClosures of 4-Isocyanato Carbon-Centered Radicals,” J. Org. Chem. 68:2960-3 (2003)) have described the synthesis of 4-bromo-1-butylaminehydrobromide by refluxing 4-amino-1-butanol in concentrated hydrobromicacid; this can be applied to prepare other bromo-alkylaminehydrobromides. As shown in Equation 3, reaction of the bromo-alkylaminehydrobromides with 2 equivalents of silver bromide in dry acetonitrilegives the nitrooxy-alklyamines (as their ammonium nitrate salts).

Treatment of the amine salts with one equivalent of imidazole in dry DMFprovides the free amine necessary for CDI coupling to SAMMA as shown inEquation 4.

Furoxan Derivatives of SAMMA.

Furoxan derivatives (furazan oxide, 1,2,5-oxadiazole 2-oxide) release NOin the presence of thiol cofactors. (Medana et al., “Furoxans as NitricOxide Donors. 4-Phenyl-3-furoxancarbonitrile: Thiol-Mediated NitricOxide Release and Biological Evaluation,” J. Med. Chem. 37: 4412-6(1994); Ferioli et al., “A new class of furoxan derivatives as NOdonors: mechanism of action and biological activity,” J. Pharmacol. 114:816-20 (1995); Schonafinger, “Heterocyclic NO prodrugs,” Farmaco. 54:316-20 (1999).) Among other therapeutic activities, furoxan derivativesinhibit H1V-1 reverse transcriptase. (Persichini, et al., “Nitric oxideinhibits the HIV-1 reverse transcriptase activity,” Biochem. Biophys.Res. Commun. 258: 624-7 (1999).) Furoxans that can be linked to SAMMAare prepared with commercially available starting materials.Nitromethane and sodium methoxide are combined in dry DMF followed bythe addition of sodium benzene sulfinate and iodine to form phenylnitromethyl sulfone (PNS). As shown in Equation 5, PNS is cyclized byheating in glacial acetic acid-nitric acid for 1 hour at 65° C. (Kelleyet al., “Synthesis of bis(arylsulfonyl)furoxans from aryl nitromethylsulfones,” J. Heterocycl. Chem. 14: 1415-6 (1977).)

The 3,4-bis(benzene sulfonyl) furoxan (BBSF) reacts with alcohols underalkaline conditions to afford substituted furoxans. (Sorba et al.,“Unsymmetrically substituted furoxans. Part 16. Reaction ofbenzenesulfonyl substituted furoxans with ethanol and ethanethiol inbasic medium,” J. Heterocycl. Chem. 33: 327-34 (1996); Loll et al, “Anew class of ibuprofen derivatives with reduced gastrotoxicity,” J. Med.Chem. 44: 3463-8 (2001)) As shown in Equation 6, treatment of BBSF with1,3-propanediol in THF with 50% NaOH leads to displacement of onesulfone group.

The resulting molecule is ready for coupling to SAMNA. Similarly,treatment of BBSF with ethanol then 1,3-propanediol forms the ethoxyderivative as indicated in Equation 7. (Sorba et al., “Unsymmetricallysubstituted furoxans. Part 16. Reaction of benzenesulfonyl substitutedfuroxans with ethanol and ethanethiol in basic medium,” J. Heterocycl.Chem. 33: 327-34 (1996).)

Another furoxan can be prepared by the reaction of crotonaldehyde withNaNO₂ in acetic acid yielding 3-methyl-4-furoxancarbaldehyde (Frutteroet al., “Unsymmetrically substituted furoxans. Part 11.Methylfuroxan-carbaldehydes,” J. Heterocycl. Chem. 26: 1345-7 (1989))which is reduced, as shown in Equation 8, to 3-methyl-4-furoxanmethanolwith NaBH₄ in dioxane. (Di Stile et al., “New 1,4-DihydropyridinesConjugated to Furoxanyl Moieties, Endowed with Both Nitric Oxide-likeand Calcium Channel Antagonist Vasodilator Activities,” J. Med. Chem.41: 5393-401 (1998).)

These can be coupled to SAMMA at different levels of substitution (e.g.,5 to 60%).

Nitrated Cellulose Sulfate.

Nitrate ester derivatives of cellulose sulfate can be prepared byregioselective sulfation of cellulose or cellulose derivative (e.g.,acetate, trimethysilyl ether, nitro, nitrite or tosylate) on C2 and G3,followed by nitration at C1, as shown below:

The reaction shown below provides good selectivity at C-6.

The amount of nitrating product can be controlled by reactiontemperature and reaction time.

The method of the present invention is carried out by applying aneffective amount of the NO-coupled anti-microbial agent or agents ofthis invention to the area or areas expected to undergo sexual contactduring the sexual activity, especially to those areas in which thetransmission of STD-causing organisms is more likely and which willlikely be in contact with a partner's bodily fluids which may containthe STD-causing organisms. For purposes of this invention, an “effectiveamount” is an amount sufficient to inactivate, but not necessarily kill,STD-causing organisms on contact and/or upon release of nitric oxide.Suitable NO-coupled anti-microbial agent or agents for use in thepresent invention include, for example, phosphorylated hesperidinscoupled with a NO-donor, sulfonated hesperidins coupled with a NO-donor,polystyrene sulfonates coupled with a NO-donor, substitutedbenzenesulfonic acid formaldehyde co-polymers coupled with a NO-donor,H₂SO₄-modified mandelic acids coupled with a NO-donor, cellulosesulfates coupled with a NO-donor, and the like. As indicated above,preferred anti-microbial agents for use in this invention includeH₂SO₄-modified mandelic acids (SAMMAs) coupled with a NO-donor (i.e.,NO-SAMMAs).

Generally, the NO-coupled anti-microbial agent or agents areincorporated into conventional carriers, such as, for example, lotions,creams, jellies, liniments, ointments, salves, oils, foams, gels,washes, suppositories, slow-releasing polymers, coatings, or devices,and the like so that they can be easily applied topically in the presentmethods. The carriers may also include other ingredients such as, forexample, pH modifiers, stabilizers, buffers, surfactants, moisturizers,colorants, thickeners, flavorings, fragrances, perfumes, and the like.The inhibitory agents of the present invention may also be used withconventional birth-control or safe-sex devices. For example, theNO-coupled anti-microbial agent or agents could be incorporated into orsimply used in conjunction with condoms (i.e., via lubricants applied tothe Interior and/or exterior surfaces), diaphragms, cervix caps, orsimilar products. The NO-coupled anti-microbial agent or agents of thepresent invention could also, for example, be released into the vagina(or rectum in the case of anal intercourse) by hand, via suppositories,or by using conventional tampon or syringe techniques. The method ofadministering or delivering the NO-coupled anti-microbial agent oragents to the potential STD-transmission site is not critical so long asan effective amount of the NO-coupled anti-microbial agent is deliveredto the site in a timely manner. Preferably the formulations and/ormethod of delivering the NO-coupled anti-microbial agent or agentsallows the inhibitory agents to remain in the appropriate area during(and even after) the sexual activity in order to maximize theeffectiveness.

Preferred inhibitory agents (i.e., the anti-microbial component of theNO-coupled anti-microbial agents) include phosphorylated hesperidins,sulfonated hesperidins, polystyrene sulfonates, substitutedbenzenesulfonic acid formaldehyde co-polymers, H₂SO₄-modified mandelicacids, and cellulose sulfates. Preferably the inhibitory agents, as wellas the NO-coupled anti-microbial agents, used are water soluble ordispersable (or at least partially so). Generally, the NO-coupledanti-microbial agents are employed at a concentration of about 0.2 mg/gor higher in a suitable formulation, preferably at a concentration ofabout 10 mg/g to about 100 mg/g, and more preferably at a concentrationof about 20 mg/g to about 70 mg/g based on the total weight of inert andactive ingredients. Although it is generally preferred that suchanti-STD compounds be used at non-cytotoxic levels in order to minimizepotential side effects, these compounds can also be used, if desired, atlevels at which the STD-organisms (or a significant portion thereof) areeffectively killed rather than simply inactivated or inhibited.

In actual use, the NO-coupled anti-microbial agent in a suitable carrieror vehicle is applied, preferably topically, to the general area orareas of expected sexual contact (e.g., areas in which bodily fluids arelikely to be generated and/or deposited) prior to the sexual activity.For vaginal heterosexual intercourse, the NO-coupled anti-microbialagents could be inserted into the vagina prior to intercourse. For analintercourse (heterosexual or homosexual), the NO-coupled anti-microbialagents could be inserted into the rectum prior to intercourse. Foreither vaginal or anal intercourse, the NO-coupled anti-microbial agentscould be Incorporated into the lubricant used with the condom. For addedprotection it is generally preferred that the NO-coupled anti-microbialagent be applied before intercourse or other sexual activity and that,if appropriate, a condom be used. For even further protection, theNO-coupled anti-microbial agents can be reapplied after completion ofthe sexual activity; in such cases, a douche or rinse with theNO-coupled anti-microbial agent in a liquid carrier solution could beused. Using edible carriers and suitable flavorings, the NO-coupledanti-microbial agents could also be used to provide protection duringoral sex (heterosexual or homosexual); a mouthwash containing NO-coupledanti-microbial agents could be used afterwards. By incorporatingdesirable flavorants, scents, fragrances, and colorants, the NO-coupledanti-microbial agents could become a “pleasing” or “desirable” componentof the sexual activity (i.e., a sex aid or toy) thereby Increasing theprobability of their use and, therefore, the degree of protectionafforded the sexual parties.

One advantage of the present method is that it can be used forprotection during a wide variety of sexual activities (vaginal, anal, ororal) by heterosexuals, bisexuals, and homosexuals of either gender.Another advantage of the present method of reducing the transmission ofSTDs is that this method can be implemented and/or used by either party.Thus, a woman could use the present method to protect herself (as wellas her partner) with or without the partners knowledge of the methodbeing used. Moreover, one partner would not be required to rely on hisor her partner's claim of being STD-free or agreement to use condoms orother barrier devices for protection. Either or both sexual partiescould initiate and Implement the use of the present method prior to, orafter, the sexual encounter. Preferably the method is used before thesexual activity and most preferably both before and after the sexualactivity. Although use only after the sexual activity would provide lessprotection, it would still be desirable to implement this methodafterwards if the method was not used prior to the sexual activity forany reason (e.g., in cases of rape). Of course, the sooner this methodis initiated after the sexual activity the better. Preferably the methodis initiated within one hour, more preferably within 15 minutes, andmost preferably almost immediately after the sexual activity. Even afterperiods greater than these, however, the use of this method as soon aspossible after the sexual activity may provide at least some protection(as compared to no treatment).

Still another advantage of the present invention is that, in contrast toother contraceptive or protective methods which rely on a cytotoxiccompound (e.g., nonoxynol-9), the NO-coupled anti-microbial agents usedin this invention do not significantly affect or inhibit the growthcharacteristics of the normal vaginal flora or otherwise significantlyirritate the vaginal tissue when used at inhibitory, noncytotoxic, orclinical concentrations. Thus, the beneficial components of normalvaginal flora are not disrupted by the use of the present invention.Significant inhibition or modifications of the vaginal flora or otherirritations (such as when nonoxynol-9 is used) can lead to increasedrisks of infections (both STD and non-STD types), unusual discharges,general discomforts, and the like, which, in turn, can lead to areluctance to use or fully take advantage of the protective method. Suchinhibition or modifications of the vaginal flora, irritation of vaginaltissue, and/or lesions can actually increase the risk of STDtransmission and infection. By avoiding or reducing the intensity ofthese effects, the present method is more likely to be used on aconsistent basis. By reducing the number of unprotected sex acts(preferably to zero) and encouraging the use of the methods of thisinvention both before and after each sex act, the overall degree ofprotection should be significantly increased. By avoiding or reducingvaginal irritations and especially lesions on the vaginal walls (orrectum lining in the case of anal intercourse), the transmission of STDshould be further reduced since transmission of STD-causing organisms isgenerally easier where damage to the cell walls has occurred. Thus,improvements in ease of use, reduction in side effects, the ability tobe initiated by either party, and the ability to be used for differentand varied sexual activities give the present invention a significantadvantage as a contraceptive and/or as an anti-STD method.

The present NO-coupled anti-microbial agents can also be used by personswho are not at risk or significant risk of pregnancy. For purposes ofthis application, the phrases “not at risk for pregnancy” or “not atsignificant risk for pregnancy” are intended to include individuals who,for any number of reasons, are not capable of becoming pregnant or whoare employing alternative birth control methods. Such individuals notcapable of becoming pregnant include, for example, homosexual partners,men in general, diagnosed sterile individuals (including womenregardless of the cause of sterility and men who are unable toimpregnate a woman regardless of the cause of sterility), post-menopausewomen, and the like. Individuals who are employing alternative birthcontrol methods, for purposes of this application, are not atsignificant risk of pregnancy. For example, a woman using acontraceptive pill and/or condom would not be considered to be at riskfor pregnancy even though the effectiveness of the pill and/or condom isnot 100 percent; similarly, users of other conventional birth controlmethods would not be considered to be “at significant risk of pregnancy”even though the failure rate may be higher than that for the pill and/orcondom. For purposes of this specification, the phrase “not at risk ofpregnancy” is also intended to also include the phrase “not atsignificant risk of pregnancy” as that term is used above.

All references (including patents, patent publications, and otherpublications) are incorporated by references in their entireties. Unlessotherwise noted, all percentages and ratios in the present specificationare based on weight.

Examples Example 1

NOT-SAMMA. This example illustrates the preparation of anti-microbialagents wherein the delivery vector is derived from SAMMA and the nitricoxide donor moiety is derived from nitrooxypropanol. In this example,the anti-microbial agent is about 7% substituted with the nitric oxidedonor moiety.

The NO-donor (3-nitrooxy-1-propanol) was synthesized by reacting silvernitrate with 3-bromo-1-propanol in acetonitrile. Silver nitrate (15.7 g,110 mmoles) was dissolved in acetonitrile (200 mL). 3-Bromo-1-propanol(18.9 g; 100 mmoles) dissolved in 25 mL acetonitrile was added. Thereaction flask was protected from light using aluminum foil. Thereaction mixture was stirred at ambient temperatures for about 96 hours;after 2 hours, a yellow precipitate (AgBr) was observed. The reactionmixture was filtered through a cellite pad to remove particulate AgBr.The solvent was removed using rotary evaporation to yield a yellow oilwhich was dissolved in dichloromethane. The resulting solution wasextracted with saturated NaCl in water to remove residual silver as AgCland then dried over anhydrous sodium sulfate. After removal of thesolvent by rotary evaporation and then distillation at about 50 mm Hg,3-nitrooxy-1-propanol, a clear yellow oil, was obtained in about 62percent yield. Identity was confirmed using IR and ¹³C NMR.

NO-SAMMA was prepared using essentially the reaction scheme shown inEquation 2 above. SAMMA (10.02 g; 74.8 acid meq; prepared as describedin Example 4 of U.S. Pat. No. 5,932,619) was dissolved in 100 mLdimethylformamide (DMF) in a flask equipped with a drying tube tomaintain low moisture levels and then cooled to 0° C. using an ice bath.A sub-stoichiometric amount of 1,1′-carbonyldiimidazole (CDI; 4.02 g;24.8 mmoles; coupling agent) was dissolved in 40 mL dry DMF and addeddropwise over 40 minutes to the stirred SAMMA solution. The reaction wascontinued for an additional 45 minutes with stirring at 0° C.Nitrooxypropanol (2.74 g; 22.6 mmoles) in 20 mL dry DMF was addeddropwise over a 30 minute period during which time the reactiontemperature was allowed to rise to ambient temperature. The reaction wascontinued for about 4 hours with stirring at ambient temperature. Thereaction mixture was decanted into 1500 mL water and then acidified topH 1.9 using 6N HCl. The resulting light pink precipitate was suctionfiltered, washed with water (500 and 250 mL portions), and suctionfiltered. The wet precipitate was dried by lyophilization to yield 10.15g of NO7-SAMMA (salmon-colored powder) in about 97 percent yield.

Example 2

NO23-SAMMA. This example illustrates the preparation of anti-microbialagents wherein the delivery vector is derived from SAMMA and the nitricoxide donor moiety is derived from nitrooxypropanol. In this example,however, the anti-microbial agent is approximately 23 percentsubstituted with the nitric oxide donor moiety.

SAMMA (3.19 g; 23.8 acid meg; prepared as described in Example 4 of U.S.Pat. No. 5,932,619) was dissolved in 500 mL dry DMF in a flask equippedwith a drying tube to maintain low moisture levels and then cooled to 0°C. using an ice bath. An excess amount of 1,1′-carbonyldiimidazole (CDI;9.73 g; 60 mmoles; coupling agent) was dissolved in 40 mL dry DMF andadded to the stirred SAMMA solution. After a further addition of 30 mLdry DMF, the reaction was continued for an additional 45 minutes withstirring at 0° C. Nitrooxypropanol (0.86 g; 7.1 mmoles; from Example 1)in 5 mL dry DMF was added dropwise over a 2 minute period. The reactiontemperature was allowed to rise to ambient temperature. The reaction wascontinued for about 15 hours with stirring at ambient temperature. Thereaction mixture was decanted into 1000 mL water and then acidified topH 1.6 using 6N HCl. The resulting deep red precipitate was suctionfiltered, washed with water (two 200 mL portions), and suction filtered.The wet precipitate was dried by lyophilization to yield 2.78 g ofNO23-SAMMA in about 73 percent yield.

Example 3

Effect of NO-SAMNA on acrosomal loss (AL). Both NO7-SAMMA (Example 1)and NO23-SAMMA (Example 2) were evaluated for their effect on acrosomalloss.

Measurement of AL was carried out as described previously. (Anderson etal., “Preclinical evaluation of sodium cellulose sulfate (Ushercell™) asa contraceptive antimicrobial agent,” J. Androl. 23: 426-38 (2002);Zaneveld et al., “Use of mandelic acid condensation polymer (SAMMA), anew antimicrobial contraceptive agent, for vaginal prophylaxis,” Fert.Steril. 78: 1107-15 ((2002).) Fresh human semen was collected fromhealthy donors by self-masturbation. All samples were used within onehour of collection. Spermatozoa were isolated and washed bycentrifugation through buffered Ficoll and resuspension in BWW medium. Asample was withdrawn for motility assessment, and the sperm suspensions'were treated with either SAMMA, 3-nitrooxypropan-1-ol, NO7-SAMMA, orNO23-SAMMA. The concentrations of SAMMA and nitrooxypropanol wereequivalent to the concentrations of these moieties found in either 0.075μg/mL NO7-SAMMA or 0.02 μg/mL NO23-SAMMA, based on their respectivedegrees of substitution. Reaction induced by 0.075 μg/mL NO7-SAMMA wascompared to the reactions induced by either 0.072 μg/mL SAMMA or 0.037μM nitrooxypropanol, and to the predicted response to the two agentsadded in combination, assuming independence of action. Similarly,reaction induced by 0.02 μg/mL NO23-SAMMA was compared to the reactionsinduced by either 0.019 μg/mL SAMMA or 0.0364 μM nitrooxypropanol. Ailreactions were carried out in either the presence or absence of addedextracellular Ga²⁺ (1.28 mM). Fifteen minutes after adding either SAMMA,nitrooxypropanol, NO7-SAMMA or NO23-SAMMA, sperm motility was measuredand spermatozoa were fixed in buffered glutaraldehyde, air-dried ontoslides, stained with Bismark Brown Y and Rose Bengal and scored for thepresence of acrosomes (De Jonge et al., “Synchronous assay for humansperm capacitation and the acrosome reaction,” J. Androl. 10: 232-39(1989)). Data are expressed as the average % maximal response, based onthe AL induced by a maximally stimulating concentration of the calciumionophore A23187.

Both NO7-SAMMA and NO23-SAMMA induced AL in the absence of addedextracellular Ca²⁺. The response to 0.075 μg/mL NO7-SAMMA in the absenceof added Ca²⁺ (41% maximal loss) was over 6-fold higher than thepredicted response to an equivalent amount of the NO donor from whichNO7-SAMMA was derived. In the presence of Ca²⁺, the response toNO7-SAMMA was synergistic over the predicted response to combinedaddition of equivalent concentrations of NO donor and SAMMA. Theincrease in AL in the presence of NO7-SAMMA, as shown in FIG. 1, wasnearly 5.6-fold than the predicted increase in NO donor-induced AL dueto the addition of SAMMA.

Even higher activity, see FIG. 1, was observed with NO23-SAMMA. Theresponse to 0.02 μg/mL NO23-SAMMA in the absence of added Ca²⁺ (about47% maximal loss) was 6.6-fold higher than the predicted response to anequivalent amount of the NO donor from which NO23-SAMMA was derived. Theobserved synergy in this instance was approximately the same as thatobserved for NO7-SAMMA, but the concentration required for the effect isonly 27% of that required for NO7-SAMMA, likely due to increased levelof substitution on NO donor. The increase in AL in the presence ofNO23-SAMMA was nearly 21.5-fold higher than the predicted increase in NOdonor-induced AL due to the addition of SAMMA. As shown in FIG. 2, theED₅₀ values of fractionated NO23-SAMMA (0.09 ng/mL: see Example 5 fordetails regarding fractionation) and unfractionated NO23-SAMMA (8 ng/mL)are more than 2800 and 30 times, respectively, less than the ED₅₀ forSAMMA (250 ng/mL).

Example 4

Effect of NO-SAMMA against C. trachomatis. Infection of HeLa cells by C.trachomatis (serotype E/UW-5/CX) was measured as described by Cooper etal. (“Chlamydia trachomatis infection of human fallopian tube organcultures,” J. Gen. Microbiol. 136: 1109-15 (1990)) in the presence andabsence of NO7-SAMMA, SAMMA, or nitrooxypropanol. Approximately 1×10⁵IFU/mL of chlamydial elementary bodies were added to differentconcentrations of either SAMMA or NO7-SAMMA, from 50 μg/mL to 500 μg/mL,and incubated at 0° C. for four hours, after which the mixture wasinoculated onto HeLa cell monolayers. In separate experiments, HeLa cellmonolayers were incubated with either SAMMA or NO7-SAMMA at differentconcentrations, ranging from 50-500 μg/mL at 37° C. for one hour, afterwhich the overlaying medium was decanted and the monolayer was washedwith fresh medium without microbicide. This was followed by inoculationof the monolayer with chlamydial elementary bodies. One hour afterinoculation, free microbes and/or microbicide were removed by washing,and the HeLa cell cultures were incubated for an additional 48 hours at37° C. Chlamydia-induced inclusions were measured by immunofluorescence,after reacting cultures with Kallsted chlamydia culture confirmationfluorescein-conjugated monoclonal antibody. Data are expressed asbacterial titer (IFU/mL) at each concentration of either SAMMA orNO7-SAMMA.

A 3-log reduction of C. trachomatis occurred at a NO7-SAMMAconcentration that is approximately one order of magnitude lower thanfor SAMMA (see FIG. 3: 3-log reduction for SAMMA alone was at 20 mg/mLas compared to 1.9 mg/mL for NO7-SAMMA). Further, the Inhibitory effectof NO7-SAMMA, unlike that of SAMMA, occurred not only at the level ofdirect effect on the elementary body, but also on the target (HeLa)cells (see FIG. 4); an IC₅₀ of 0.7 mg/mL was found for NO7-SAMMA.Interestingly, the inhibitory effect of nitroprusside against C.trachomatis (IC₅₀=23 μM) was about the same as that against spermatozoa(74% maximal AL at 22 μM), suggesting that C. trachomatis andspermatozoa may have similar sensitivity to NO.

Example 5

Fractioned NO23-SAMMA. This example illustrates the preparation andevaluation of an anti-microbial agent, wherein the delivery vector isderived from SAMMA that has been fractionated on silica gel to achieve amore narrow range of molecular weights, and the nitric oxide donormoiety is derived from nitrooxypropanol. This fractionated material isdistinguished from that described in Example 2 wherein non-fractionated(bulk) SAMMA was used as the delivery vector. In this example, theanti-microbial agent is approximately 23 percent substituted with thenitric oxide donor moiety. This fractionated material exhibitedunexpectedly increased activity as compared to the unfractionatedmaterial.

SAMMA (3.0 g; 22.4 acid meq, prepared as described in Example 4 of U.S.Pat. No. 5,932,619) was dissolved in a minimal volume (9 mL) ofmethanol. This solution was applied to a 200×35 mm glass column filled ⅔with chromatographic silica gel, 100-200 mesh (Fisher Scientific),equilibrated with methylene chloride and topped with washed sand, andeluted (200 mL each) with a discontinuous gradient of methylene chloridecontaining increasing concentrations of methanol (2%, 10%, 20%, 30%100%, v/v; increasing polarity). Fractions of 200 mL were collected.Fractionated SAMMA (to be used for the preparation of fractionatedNO23-SAMMA) was recovered in the 30% methanol (20-30% fraction) elution.Yield: 2.55 g (85%). MALDI TOF MS showed a predominant molecular weightdistribution of the fractionated SAMMA between 700-2500, with molecularweight <600 representing a minor (approx. 2-3%) constituent, and verylow (<1%) amounts with molecular weight equal to or greater than 2500.The 20-30% fraction was used to prepare fractionated NO23-SAMMA usingthe procedure described in Example 2 for NO23-SAMMA.

Fractionated NO23-SAMMA has the highest activity as a stimulus ofacrosomal loss of any the compounds studied (see FIG. 2). Covalentmodification of SAMMA having a more dearly defined range of molecularweights increased efficacy by about two orders of magnitude overNO23-SAMMA. SAMMA fractionated on silica gel is similar tounfractionated (bulk) SAMMA as a stimulus of AL (68% maximal AL at 0.25μg/mL; essentially the same ED₅₀ as for unfractionated SAMMA). Althoughthis represents a synergistic response to equivalent concentrations ofeither nitrooxypropanol or SAMMA added alone (separately), synergismcannot be quantified for fractionated NO23-SAMMA, since equivalentconcentrations of SAMMA and NO donor are so low as to produce responsesbelow the limit of detection. The ED₅₀ of fractionated NO23-SAMMA is0.09 ng/mL. This quantity of fractionated NO23-SAMMA contains theequivalent of 0.08 ng/mL SAMMA and 0.13 nM nitrooxypropanol; it,however, represents an increased activity over SAMMA of nearly2,800-fold. For comparison, the ED₅₀ of NO23-SAMMA (8 ng/mL) containsthe equivalent of 6.8 ng/mL SAMMA and 11.2 nM nitrooxypropanol. The ED₅₀for nitrooxypropanol as a stimulus of acrosomal loss is 120 nM.

Fractionated NO23-SAMMA has essentially no effect (i.e., less than about10% inhibition) on the percentage of motile spermatozoa atconcentrations up to 10 mg/mL (Control motility=69.4±0.6 (SEM) %;motility with 10 mg/mL fractionated NO23-SAMMA=63.2±2.2%; N=4). Thesedata show that NO-SAMMA has no effect on sperm viability.

Acrosomal loss induced by SAMMA is Ca²⁺-dependent (Anderson et al.,“SAMMA induces premature acrosomal loss by Ca²⁺ signalingdysregulation”, J. Andra 27: 568-577 (2006)). Unless otherwise noted,acrosomal loss data were obtained from assays that included Ca²⁺ in theextracellular medium. In contrast, acrosomal loss induced by NO donorsoccurs independent of Ca²⁺. These properties can be exploited todetermine the contributions of the SAMMA and NO donor moieties toacrosomal loss induced by NO-SAMMA. Although not wishing to be limitedby theory, it appears that acrosomal loss in the presence of addedextracellular Ca²⁺ may be due to either or both moieties, whereasacrosomal loss in the absence of Ca²⁺ is due entirely to the NO donormoiety.

Fractionated NO23-SAMMA induces acrosomal loss in the absence of Ca²⁺with an ED₅₀ of 0.37 ng/mL. Based on nitrogen content of fractionatedNO23-SAMMA (1.98±0.106%), this is equivalent to 0.53 nM equivalents ofnitric oxide donor (see FIG. 5). SAMMA at 0.25 μg/mL in the absence ofCa²⁺ has essentially no effect (Anderson et al., “SAMMA inducespremature acrosomal loss by Ca²⁺ signaling dysregulation”, J. Androl.27: 568-577 (2006)), and the ED₅₀ for nitrooxypropanol is 0.12 μM(Ca²⁺-independent). The effect is of fractionated NO23-SAMMA in theabsence of Ca²⁺ is likely due to the NO donor moiety of NO-SAMMA, and isclearly enhanced relative to effects of either SAMMA or NO donor addedalone. Strictly speaking, this does not represent a synergisticresponse, since SAMMA is without effect in the absence of Ca²⁺.

Contraception in rabbits by fractionated NO23-SAMMA is substantiallymore effective than contraception by SAMMA. Greater efficacy is seen ata fractionated NO-SAMMA concentration one order of magnitude lower thanthe concentration of SAMMA. Sperm pretreatment with 5 mg/mL SAMMAreduces fertilization. However, contraception of SAMMA is incompletewith 0.5 mg/mL SAMMA being essentially without effect. In contrast, 0.5mg/mL fractionated NO23-SAMMA is essentially completely contraceptive;only 1 of 120 oocytes examined from 5 rabbits was fertilized. Thecontraceptive data, reported below, were obtained using washed rabbitspermatozoa incubated with the test compounds for about 15 minutes at37° C. prior to insemination. About 22 to 34 million spermatozoa wereused for fertilization testing. Oocytes were harvested about 25 to 27hours post insemination and scored for fertilization. Averagefertilization percentages per rabbit (along with 90% confidence limits)were as follows:

Oocytes Examined % Fertilization Test Compound (No. Of Rabbits Used)(90% confidence limits)* None (control)  269 (12) 90 (75.2-98.7)^(A)SAMMA  79 (3) 78 (29.0-99.7)^(A) (0.5 mg/mL) SAMMA 182 (7) 7(1.2-18.2)^(B) (5 mg/mL) Fractionated 120 (5) 0.7 (0-6.8)^(C) NO23-SAMMA(0.5 mg/mL) *Values with different superscript letter values aresignificantly different using Newman-Keuls multiple range test. ^(A) and^(B) values are different at a p value < 0.001; ^(B) and ^(C) values aredifferent at a p value of 0.055.

NO23-SAMMA, whether fractionated or not, appears, at least in part, toact through release of nitric oxide. Fractionated NO23-SAMMA-inducedacrosomal loss in the absence of added extracellular Ca²⁺ is inhibitedby the selective protein kinase G inhibitor KT5823 (see FIG. 6). Asnoted above, SAMMA-induced acrosomal loss (SAL) is also inhibited byKT5823, as well as by nitric oxide synthase and guanylate cyclaseinhibitors, suggesting a role of NO via the cGMP/protein kinase Gpathway in this process. Acrosomal loss in human spermatozoa in responseto NO donors is inhibited by protein kinase G inhibitors (Revelli etal., “Signaling pathway of nitric oxide-induced acrosome reaction inhuman spermatozoa”, Biol. Reprod., 64: 1708-12 (2001)). SAMMA isineffective in inducing acrosomal loss in the absence of addedextracellular Ca²⁺. By inference, acrosomal loss induced by fractionatedNO23-SAMMA in the absence of added extracellular Ca²⁺ is likely mediatedby NO release from the NO donor moiety of fractionated NO23-SAMMA.Inhibition of acrosomal loss by the selective protein kinase G inhibitorsupports this contention. The IC₅₀ for inhibition of fractionatedNO23-SAMMA-induced acrosomal loss (Ca^(a+) independent) by KT5823 is 0.7μM (FIG. 6).

Fractionated NO23-SAMMA has activity against HIV and HSV. When directcomparisons are made, IC₅₀ values for NO-SAMMA are slightly higher thanthose for SAMMA. However, concentrations of fractionated NO-SAMMArequired for 3-Log reduction in infectivity are lower than those forSAMMA. These results suggest that at higher concentrations, fractionatedNO-SAMMA is more effective than SAMMA against HIV and HSV; the change inrelative efficacy may reflect the contribution of nitric oxide releaseagainst these pathogens. HIV is sensitive to inhibition by the NO donorused to synthesize NO-SAMMA (nitrooxypropanol), although substantiallyless than to NO-SAMMA. Experiments were conducted to compare theactivities of SAMMA and fractionated NO23-SAMMA against HIV-1 BaLinfected primary lymphocytes. Host cells were Inoculated with virus (200TCID50/2×1⁰⁵ cells) for 2 h, and washed to remove unbound virus, beforeeither SAMMA or fractionated NO23-SAMMA was added to the cultures. Viralreplication was measured on day 7 of incubation (p24 levels). In allinstances, viability of the target cells remained at approximately 98%for the duration of the experiments. These data are presented below.

IC₅₀ 3-Log reduction Compound (μg/mL) (μg/mL) SAMMA 66 6560 Fractionated208 2084 NO23-SAMMA Nitrooxypropanol 2544 1017 (80% reduction)

SAMMA and fractionated NO23-SAMMA are highly active in preventinginfection of lymphocytes by HIV-1 BaL. IC₅₀ values are less than 10μg/mL. Fractionated NO23-SAMMA concentrations required to inhibit p24values by 50% and 3-logs are about 3-fold lower than those for SAMMA.The dose-response for fractionated NO23-SAMMA between 1 μg/mL and 10μg/mL is more responsive than that for SAMMA, suggesting involvement ofthe NO-SAMMA NO donor moiety. This possibility is likely, in view ofpossible reduced binding affinity of NO-SAMMA relative to SAMMA, due toreduced charge density of the NO donor adduct.

The ability of fractionated NO23-SAMMA to reduce infectivity of HSV-1(F) and HSV-2 (0) was compared with the anti-HSV activities of theparent compound, SAMMA. Both agents are highly effective against theselaboratory strains. In contrast to results obtained for HIV-1, IC₅₀values are somewhat lower for SAMMA than for fractionated NO23-SAMMA.However, fractionated NO23-SAMMA is more effective in nearly completelyinhibiting both viruses; 3-Log reductions are observed at NO-SAMMAconcentrations that are 80% to 87% lower than SAMMA concentrationsrequired for the same effect. Dose-responses for fractionated NO23-SAMMAare thus delayed, but sharper as compared with SAMMA. The slightlyincreased IC₅₀ values for fractionated NO23-SAMMA suggest increasedsensitivity of HSV binding to changes in charge density caused by thecovalent attachment of the NO donor moiety.

HSV Studies: One hour after adding serial 2-fold dilutions of eitherSAMMA or fractionated NO23-SAMMA to confluent human fibroblasts(foreskin) at concentrations ranging from 2 μg/mL to 256 μg/mL, cellswere inoculated with either HSV-1 (F) or HSV-2 (G) (ATCC; MOI=0.05).After 48 hours (35° C., 5% CO₂), cells were visually examined for viralcytopathogenic effect (CPE) in the virus control wells. Cells were fixedand blocked (PBS with 0.2% BSA and 0.05% Tween 20). Viral titers weredetermined by ELISA, with HRP-conjugated polyclonal antibodies (DakoCorp, Carpinteria, Calif.). Reaction with 3,3′,5,5′-tetramethylbenzidine(TMB) was measured spectrophotometrically (630 nm and 450 am). Data wereexpressed as mean percentage of control viral incubations (±SEM) towhich no microbicide was added.

HIV Studies: Primary lymphocytes and virus were incubated withmicrobicide (serial 10-fold dilutions, ranging from 1 ng/mL to 1 mg/mL)for 1 hour, followed by inoculation (50 TCID50/2×10⁵ cells). After 2hours, cells were washed to remove virus. Incubations continued withmicrobicide for 7 days, after which viral titers (p24) were measured.Data (μg/well) were expressed as mean±SEM of triplicate determinations.The data are consistent with activities of SAMMA and fractionatedNO23-SAMMA in HIV-1 BaL-infected lymphocytes and suggest a greatercontribution of the NO donor moiety of fractionated NO23-SAMMA at higherconcentrations.

These data for both the HSV and HIV studies are presented below. Thevalues for each dose-response curve includes the coefficient ofdetermination (r²), degrees of freedom (DoF), calculated concentrationof inhibitor required to reduce viral titer by 50 percent (IC₅₀), andconcentration of inhibitor to reduce viral titer by 99.9 percent(3-log).

SAMMA Fractionated NO23-SAMMA r² IC₅₀ 3-log r² IC₅₀ 3-log (DoF) (μg/mL)(μg/mL) (DoF) (μg/mL) (μg/mL) HIV-1 0.9999 6.5 60 0.9999 2.5 23 BaL(6)    (6)    HSV-1 0.9993 7.8 214 1.0000 22 42 (F) (6)    (6)    HSV-20.994  1.5 181 0.994  5.2 24 (G) (9)    (7)   

Example 6

NO-SAMMA retains many biological properties of the parent compound,SAMMA, including the ability to inhibit hyaluronidase (a propertybelieved to be responsible, in part, for some anti-microbial activity),and lack of effects on sperm motility and growth of lactobacilli(indicators of specificity of action and lack of general cytotoxiceffects on spermatozoa and beneficial vaginal flora. See generally,Zaneveld et al. “Method for preventing sexually transmitted diseases,”U.S. Pat. No. 5,932,619. This example evaluates some of these propertiesfor fractionated NO23-SAMMA (prepared as in Example 5).

Hyaluronidase activity was measured as described in Example 8 of U.S.Pat. No. 5,932,619. Activity of fractionated NO23-SAMMA againsthyaluronidase is very similar to that of SAMMA. In contrast, the NOdonor moiety of NO23-SAMMA, nitrooxypropanol, is nearly without effect.The following results were obtained.

3-Log IC₅₀ reduction* Inhibition ± SEM Agent (μg/mL) (μg/mL) (N = 4)Fractionated 11.1 13.6 100 ± 2.0% at 15 μg/mL ** NO23-SAMMA SAMMA 8.114.9 104 ± 2.0% at 15 μg/mL Nitrooxypropanol — — 8 ± 1.8% at 50 μM*concentration required for 99.9% inhibition of activity ** 15 μg/mLfractionated NO23-SAMMA contains 21.2 μM equivalent of nitrooxypropanol

Sperm immobilization by fractionated NO23-SAMMA was evaluated by amodification (Anderson et al. “Evaluation of poly(styrene-4-sulfonate)as a preventive agent for conception and sexually transmitted diseases,”J Androl 21:862-875 (2000)) of the method of Sander and Cramer (“Apractical method for testing the spermicidal action of chemicalcontraceptives,” Hum Fertil 6:134-137, 153 (1941)). Thirty seconds afteradding different concentrations of the test agent (2.5 to 20 mg/mL forSAMMA and fractionated NO23-SAMMA and 1-10 mM for nitrooxypropanol), thefraction of motile spermatozoa was determined with brightfieldmicroscopy (400×). Data are presented as the percentage of motilespermatozoa at each concentration of test agent. When possible, testoutcomes were also reported as the concentration of agent in the semensample that reduces motility by 50%.

Neither fractionated NO23-SAMMA, SAMMA, nor nitrooxypropanol can beregarded as spermicidal. Sperm motility is reduced by less than 10percent by concentrations of these agents that are 4-8 orders ofmagnitude greater than concentrations required to induce acrosomal loss.The data are as follows.

% Motile Spermatozoa (at 10 mg/mL test agent) Test Agent av ± SEM (n =4) IC₅₀ None 70 ± 0.6 — Fractionated NO23-SAMMA 63 ± 2.2 at 10 mg/mL 30mg/mL SAMMA 67 ± 0.6 at 10 mg/mL 64 mg/mL Nitrooxypropanol 66 ± 0.5 at10 mM 163 mM

The effect of fractionated NO23-SAMMA on growth of L. gasseri wasdetermined as described in Example 9 of U.S. Pat. No. 5,932,619. Similarto SAMMA, fractionated NO23-SAMMA has no effect on lactobacillus growthat concentrations up to 10 mg/mL. These data are shown below.

Fractionated Doubling Difference NO23-SAMMA Time from Control (mg/mL)(minutes) (Confidence Level) 0 121.1 — 5.0 125.4 >0.1 10.0 130.8 >0.1

The release of nitric oxide from NO-SAMMA has been confirmed. Althoughavailable instrumentation lacked sensitivity to detect NO release in thebiological systems that have been tested, chemical-induced release of NOfrom relatively high concentrations of NO-SAMMA could be quantified.

NO formation was measured by a modification of the method of Bertlnariaet al. (“Synthesis and anti-Helicobacter pylori properties ofNO-donor/metronidazole hybrids and related compounds,” Drug Devel. Res.60: 225-39, (2003). Fractionated NO23-SAMMA (0.1 mg/mL) was reacted with50 mM cysteine in 50 mM sodium phosphate (pH 7.4 at 37° C.) for up to 24hours. Equal volumes of reaction mixture and Griess reagent were reactedfor 15 minutes and the absorbency at 540 nm was determined. Nitritestandards produced a linear standard curve (r²=0.9999) in the range 1-50μM, from which nitrite formation from nitrooxypropanol was measured.Higher concentrations could not be evaluated, since the pink/redreaction product when combined with the Griess reagent precipitated andcould not be measured.

Nitrite formation (an indirect measure of NO formation) fromfractionated NO23-SAMMA increased over time. The results are presentedin FIG. 7. Nitrite formation when fitted to a kinetic curve showed firstorder sequential formation, A→B→C, which is similar to that describingnitrite formation from nitrooxypropanol.

The embodiments and examples described and discussed above are intendedto illustrate the present invention and not to limit the scope of theinvention which is defined in the appended claims.

1-16. (canceled)
 17. An anti-microbial agent for reducing risk oftransmitting a sexual transmitted disease, said anti-microbial agentcomprising a covalent adduct of a delivery vector having anti-microbialactivity and a nitric oxide donor, the nitric acid donor consisting of anitric acid moiety and a spacer, the nitric acid donor being covalentlybonded through the spacer to the delivery vector having anti-microbialactivity, wherein NO is released from the anti-microbial agent duringuse.
 18. The anti-microbial agent of claim 17, wherein the deliveryvector has contraceptive activity and anti-microbial activity.
 19. Theanti-microbial agent as defined in claim 17, wherein the delivery vectoris selected from the group consisting of phosphorylated hesperidins,sulfonated hesperidins, polystyrene sulfonates, substitutedbenzenesulfonic acid formaldehyde co-polymers, H₂SO₄-modified mandelicacids, and cellulose sulfates.
 20. The anti-microbial agent as definedin claim 17, wherein the NO-donor is selected from the group consistingof nitrate esters, furoxans, ketoximes, S-nitrosothiols,nitrosohydrazines, and hydroxylamides.
 21. The anti-microbial agent asdefined in claim 19, wherein the NO-donor is selected from the groupconsisting of nitrate esters, furoxans, ketoximes, S-nitrosothiols,nitrosohydrazines, and hydroxylamides.
 22. The anti-microbial agent asdefined in claim 19, wherein the delivery vector is a H2SO4-modifiedmandelic acid.
 23. The anti-microbial agent as defined in claim 17,wherein the NO-donor is a nitrate ester.
 24. The anti-microbial agent asdefined in claim 17, wherein the anti-microbial agent is contained in aninert carrier.
 25. The anti-microbial agent as defined in claim 18,wherein the anti-microbial agent is contained in an inert carrier. 26.The anti-microbial agent as defined in claim 25, wherein theanti-microbial agent is at a concentration greater than about 0.2 mg/g.27. The anti-microbial agent as defined in claim 26, wherein theanti-microbial agent is at a concentration greater than about 0.2 mg/g.28. The anti-microbial agent as defined in claim 26, wherein theanti-microbial agent is at a concentration of about 10 to 100 mg/g. 29.The anti-microbial agent as defined in claim 27, wherein theanti-microbial agent is at a concentration of about 10 to 100 mg/g. 30.A method for reducing the risk of transmission and infection by asexually transmitted disease through sexual activity between two or moreparties, said method comprising: applying an effective amount of ananti-microbial agent to an area of the body to be engaged in the sexualactivity of at least one of the parties prior to the sexual activity andthen engaging in the sexual activity, wherein the antimicrobial agentcomprises: a covalent adduct of a delivery vector having anti-microbialactivity and a nitric oxide donor, the nitric acid donor consisting of anitric acid moiety and a spacer, the nitric acid donor being covalentlybonded through the spacer to the delivery vector having anti-microbialactivity, wherein NO is released from the anti-microbial agent afterapplying to the area of the body.